Angiotensin converting enzyme homolog and uses therefor

ABSTRACT

The present invention relates to the discovery of novel genes encoding an angiotensin converting enzyme, Angiotensin Converting Enzyme-2 (ACE-2). The invention provides therapeutics, prognostic and diagnostics methods for treating blood pressure related disorders as well as various types of allergic conditions, among others. Also disclosed are screening assays for identifying compounds for treating and preventing these conditions.

RELATED APPLICATIONS

This application claims priority to U.S. application Ser. No.08/989,299, filed on Dec. 11, 1997, U.S. application Ser. No.09/163,648, filed on Sep. 30, 1998, U.S. application Ser. No.09/407,427, filed on Sep. 29, 1999, and PCT Patent Application No.:PCT/US99/22976, filed on Sep. 29, 1999, incorporated herein in theirentirety by this reference.

SUMMARY OF THE INVENTION

Hypertension, or high blood pressure, is the most common diseaseaffecting the heart and blood vessels. Statistics indicate thathypertension occurs in more than 50 million Americans. The prevalence ofhypertension increases with age. Between 85 and 90% of cases are primary(i.e., essential) hypertension, i.e., a persistently elevated bloodpressure that cannot be attributed to any particular organic cause. Theremaining percentage of cases are secondary hypertension, i.e., elevatedblood pressure having an identifiable underlying cause such as kidneydisease and adrenal hypersecretion.

Hypertension is of considerable concern because of the harm it can do tothe heart, brain, and kidneys if it remains uncontrolled. The heart ismost commonly affected by high blood pressure. When blood pressure ishigh, the heart uses more energy in pumping against the increasedresistance caused by the elevated arterial blood pressure. Because ofthe increased effort, the heart muscle thickens and the heart becomesenlarged and needs more oxygen. If it cannot meet the demands put on it,angina pectoris or even myocardial infarction may develop. Hypertensioncan result in numerous complications include left ventricular failure;atherosclerotic heart disease; retinal hermorrhages, exudates,papilledema, and vascular accidents; cerebrovascular insufficiency withor without stroke; and renal failure. An untreated hypertensive patientis at great risk of developing disabling or fatal left ventricularfailure, myocardial infarction, cerebral hemorrhage or infarction, orrenal failure at early age. Hypertension is the most important riskfactor predisposing to stroke and is an important risk factorpredisposing to coronary atherosclerosis.

An abnormal blood pressure can also result from specific conditions ordiseases, such as heart failure. Heart failure is a chronic or acutestate that results when the heart is not capable of providing sufficientcardiac output to satisfy the metabolic needs of the body. Heart failureis commonly referred to as congestive heart failure (CHF), sincesymptoms of increased venous pressure (pulmonary congestion with leftheart failure and peripheral edema with right heart failure) are oftenpredominant. Symptoms and signs of CHF include fatigue, peripheral andpulmonary edema, and visceral congestion (e.g., dyspnea). These symptomsare produced by diminished blood flow to the various tissues of the bodyand by accumulation of excess blood in the various organs, that resultsfrom the heart being incapable of pumping out the blood. Heart failurecan result from several underlying diseases, most commonly inindustrialized nations from atherosclerotic coronary artery disease withmyocardial infarction. Myocardidis, various cardiomyopathies, andvalvular and congenital defects may also result in heart failure(Anderoli et al., Cecil: Essentials of Medicine, Third Edition, WBSaunders Company, 1993). A major problem in CHF is the inability of thefailing left ventricle to maintain a normal blood pressure, thusresulting in increased pre- and afterload, and leading to progressiveventricular dilation with wall remodeling. Vasodilators which induce areduction in pre- and afterload, i.e., reduction of the systemicvascular resistance and reduction of the peripheral vascular resistance,respectively, are currently used to treat CHF (Lionel H. Opie, Drugs forthe Heart, Third Edition, WB Saunders Company, 1991).

One important system involved in regulating blood pressure is therenin-angiotensin-aldosterone system. In this system, renin, aproteolytic enzyme formed in the granules of the juxtaglomerularapparatus cells catalyzes the conversion of angiotensinogen (a plasmaprotein) into angiotensin I, a decapeptide. This inactive product isthen cleaved by a converting enzyme, termed angiotensin convertingenzyme (ACE) mainly in the lung, but also in the kidney and brain, to anoctapeptide, angiotensin II, which is a potent vasoconstrictor and alsostimulates the release of aldosterone. Aldosterone is an adrenal cortexhormone that promotes the retention of salt and water by the kidneys andthus increases plasma volume, resulting in an increase in bloodpressure. Angiotensin II also stimulates the release of norepinephrinefrom neural cells which interacts with specific receptors on bloodvessels, thereby resulting in an increase in calcium andvasocontriction. Another mechanism by which angiotensin II inducesvasoconstriction is by interacting with specific receptors on bloodvessels, thereby resulting in an opening of calcium channels and anincrease in calcium, resulting in vasoconstriction.

ACE, also referred to as peptidyl dipeptidase A (EC 3.4.15.1) andkininase II is a metallopeptidase, more particularly a zinc peptidasewhich hydrolyses angiotensin I and other biologically activepolypeptides, such as kinins, e.g., bradykinin. Bradykinin is avasodilator, which acts at least in part by inducing release ofvasodilator prostaglandins, and which is inactivated upon hydrolysis byACE. Thus, ACE increases blood pressure at least in part by producingangiotensin II, a vasoconstrictor, and by inactivating bradykinin, avasodilator. Bradykinin is also involved in other biological activitiesincluding mediation of pain and inflammatory reactions.

The role of ACE in regulating blood pressure is further demonstrated atleast by the efficacy of ACE inhibitors in reducing hypertension andtreating CHF in individuals. ACE inhibitors have major roles asvasodilators in hypertension and CHF and are among the most efficientdrugs for treating these disorders (see, e.g., Opie et al., AngiotensinConverting Enzyme Inhibitors and Conventional Vasodilators, in Lionel H.Opie, Drugs for the Heart, Third Edition, WB Saunders Company, 1991,p106). Several clinical trials indicate that ACE inhibitors prolongsurvival in a broad spectrum of patients with myocardial infarction andheart failure, ranging from those who are asymptomatic with ventriculardysfunction to those who have symptomatic heart failure but arenormotensive and hemodynamically stable. For example, one studydemonstrated a 40% reduction in mortality at 6 months in patients withsevere heart failure (The CONSENSUS Trial Study Group, N. Engl. J. Med.316:1429 (1987); The CONSENSUS Trial Study Group, N. Engl. J. Med.325:293 (1991)).

ACE cleaves substrates other than angiotensin I and bradykinin. Forexample, ACE cleaves enkephalins, as well as heptapeptide andoctapeptide enkephalin precursors. ACE also hydrolyzes thetridecapeptide neurotensin to a dipeptide and undecapeptide (Skidgel etal. In Neuropeptides and Their Peptidases, Ed. Turner A J, Chichester, UK, Ellis-Horwood, (1987)). ACE can also cleave and thereby inactivatesubstance P (Skidgel et al., supra).

Several ACE inhibitors are currently available on the market (e.g.,Captopril, Enalapril, Fosinopril, Lisinopril, and Ramipril). However,ACE inhibitors in large doses can cause a variety of undesirablesecondary effects including nephrotic syndrome, membraneousglomerulonephritis, nephritis, and leukopenia, as well as angioedema.

The isolation of novel nucleic acids coding novel ACE proteins would beuseful, e.g., in developing drugs which are capable of regulating theactivity of ACE without having the negative secondary effects.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery of anovel gene encoding a novel human protein, having sequence homologieswith known angiotensin converting enzymes (ACEs). Thus, the newlyidentified proteins and nucleic acids described herein are referred toas “angiotensin converting enzyme-2” or “ACE-2”. The human ACE-2 genetranscript is shown in FIG. 1 (SEQ ID NO:1) and includes 5′ and 3′untranslated regions and a 2415 base pair open reading frame (SEQ IDNO:3) encoding an 805 amino acid polypeptide having SEQ ID NO:2. Themature protein, i.e., the full length protein without the signalsequence is comprised of about 787 amino acids. ACE-2 is expressedpredominantly in kidney and testis. A nucleic acid comprising the cDNAencoding the full length human ACE-2 polypeptide has been deposited atthe American Type Culture Collection (12301 Parklawn Drive, Rockville,Md.) on Dec. 3, 1997 has been assigned ATCC Designation No. 209510.

An amino acid and nucleotide sequence analysis using the BLAST program(Altschul et al. (1990) J. Mol. Biol. 215:403) revealed that certainportions of the amino acid and nucleic acid sequences of the newlyidentified human ACE-2 protein and nucleic acid have a sequencesimilarity with certain regions of angiotensin converting enzymes. Inparticular, the amino acid sequence of the zinc binding domain, which isconserved in all ACE proteins identified to date and which is located inthe catalytic site of the enzyme and necessary for catalytic activity,is also found in ACE-2. Amino acids which have been identified as eithercontacting the zinc atom and/or involved in the catalysis and areconserved among all ACE proteins, are present in ACE-2. Thus, ACE-2 isbelieved to share at least some of the biological activities of ACEproteins, in particular the peptidase activity. In fact, as shown herein(see Example 5.4), ACE-2 cleaves the C-terminal amino acid fromangiotensin I to produce Ang (1-9). ACE-2 also comprises a transmembranedomain which is present in most ACE proteins and which is likely tomediate protein attachment to the cell membrane. Except for the presenceof other small regions of homology between ACE-2 and known ACE proteins,the other portions of ACE-2 are significantly different from those ofknown ACE proteins.

In one aspect, the invention features isolated ACE-2 nucleic acidmolecules. In one embodiment, the ACE-2 nucleic acid is from avertebrate. In a preferred embodiment, the ACE-2 nucleic acid is from amammal, e.g. a human. In an even more preferred embodiment, the nucleicacid has the nucleic acid sequence set forth in SEQ ID NO:1 and/or 3 ora portion thereof. The disclosed molecules can be non-coding, (e.g. aprobe, antisense, or ribozyme molecules) or can encode a functionalACE-2 polypeptide (e.g. a polypeptide which specifically modulatesbiological activity, by acting as either an agonist or antagonist of atleast one bioactivity of the human ACE-2 polypeptide). In oneembodiment, the nucleic acid molecules can hybridize to the ACE-2 genecontained in ATCC designation No. 209510. In another embodiment, thenucleic acids of the present invention can hybridize to a vertebrateACE-2 gene or to the complement of a vertebrate ACE-2 gene. In a furtherembodiment, the claimed nucleic acid can hybridize with a nucleic acidsequence shown in FIG. 1 (SEQ ID NOs: 1 and 3) or complement thereof. Ina preferred embodiment, the hybridization is conducted under mildlystringent or stringent conditions.

In further embodiments, the nucleic acid molecule is an ACE-2 nucleicacid that is at least about 70%, preferably about 80%, more preferablyabout 85%, and even more preferably at least about 90% or 95% homologousto the nucleic acid shown as SEQ ID NOs: 1 or 3 or to the complement ofthe nucleic acid shown as SEQ ID NOs: 1 or 3. In a further embodiment,the nucleic acid molecule is an ACE-2 nucleic acid that is at leastabout 70%, preferably at least about 80%, more preferably at least about85% and even more preferably at least about 90% or 95% similar insequence to the ACE-2 nucleic acid contained in ATCC designation No.209510 or shown set forth in SEQ ID NOs: 1 and/or 3 or complementthereof.

The invention also provides probes and primers comprising substantiallypurified oligonucleotides, which correspond to a region of nucleotidesequence which hybridizes to at least about 6 at least about 10, and atleast about 15, at least about 20, or preferably at least about 25consecutive nucleotides of the sequence set forth as SEQ ID NO:1 orcomplements of the sequence set forth as SEQ ID NO:1 or naturallyoccurring mutants or allelic variants thereof, such as those describedin the Examples. In preferred embodiments, the probe/primer furtherincludes a label group attached thereto, which is capable of beingdetected.

For expression, the subject nucleic acids can be operably linked to atranscriptional regulatory sequence, e.g., at least one of atranscriptional-promoter (e.g., for constitutive expression or inducibleexpression) or transcriptional enhancer sequence. Such regulatorysequences in conjunction with an ACE-2 nucleic acid molecule can providea useful vector for gene expression. This invention also describes hostcells transfected with said expression vector whether prokaryotic oreukaryotic and in vitro (e.g. cell culture) and in vivo (e.g.transgenic) methods for producing ACE-2 proteins by employing saidexpression vectors.

In another aspect, the invention features isolated ACE-2 polypeptides,preferably substantially pure preparations, e.g. of plasma purified orrecombinantly produced polypeptides. The ACE-2 polypeptide can comprisea full length protein or can comprise smaller fragments corresponding toone or more particular motifs/domains, or fragments comprising at leastabout 5, 10, 25, 50, 75, 100, 125, 130, 135, 140 or 145 amino acids inlength. In particularly preferred embodiments, the subject polypeptidehas an ACE-2 bioactivity, for example, it is capable of interacting withand/or hydrolyzing a target peptide, such as angiotensin I, kinetensin,bradykinin or neurotensin.

In a preferred embodiment, the polypeptide is encoded by a nucleic acidwhich hybridizes with the nucleic acid sequence represented in SEQ IDNOs: 1 and 3. In a further preferred embodiment, the ACE-2 polypeptideis comprised of the amino acid sequence set forth in SEQ ID NO:2. Thesubject ACE-2 protein also includes within its scope modified proteins,e.g. proteins which are resistant to post-translational modification,for example, due to mutations which alter modification sites (such astyrosine, threonine, serine or aspargine residues), or which preventglycosylation of the protein, or which prevent interaction of theprotein with intracellular proteins involved in signal transduction.

The ACE-2 polypeptides of the present invention can be glycosylated, orconversely, by choice of the expression system or by modification of theprotein sequence to preclude glycosylation, reduced carbohydrate analogscan also be provided. Glycosylated forms can be obtained based onderivatization with glycosaminoglycan chains. Also, ACE-2 polypeptidescan be generated which lack an endogenous signal sequence (though thisis typically cleaved off even if present in the pro-form of theprotein).

In yet another preferred embodiment, the invention features a purifiedor recombinant polypeptide, which has the ability to modulate, e.g.,mimic or antagonize, an activity of a wild-type ACE-2 protein, e.g., itsability to bind and/or hydrolyze angiotensin I, kinetensin, bradykinin,or neurotensin, or a peptide having a significant amino acid homologythereto. Preferably, the polypeptide comprises an amino acid sequenceidentical or homologous to a sequence designated in SEQ ID No: 2.

Another aspect of the invention features chimeric molecules (e.g.,fusion proteins) comprising an ACE-2 protein. For instance, the ACE-2protein can be provided as a recombinant fusion protein which includes asecond polypeptide portion, e.g., a second polypeptide having an aminoacid sequence unrelated (heterologous) to the ACE-2 polypeptide. Apreferred ACE-2 fusion protein is an immunoglobulin-ACE-2 fusionprotein, in which an immunoglobulin constant region is fused to an ACE-2polypeptide.

Yet another aspect of the present invention concerns an immunogencomprising an ACE-2 polypeptide in an immunogenic preparation, theimmunogen being capable of eliciting an immune response specific for anACE-2 polypeptide; e.g. a humoral response, an antibody response and/orcellular response. In a preferred embodiment, the immunogen comprises anantigenic determinant, e.g. a unique determinant of a protein encoded bythe nucleic acid set forth in SEQ ID NO:1 or 3; or as set forth in SEQID NO:2.

A still further aspect of the present invention features antibodies andantibody preparations specifically reactive with an epitope of an ACE-2protein.

The invention also features transgenic non-human animals which include(and preferably express) a heterologous form of an ACE-2 gene describedherein, or which misexpress an endogenous ACE-2 gene (e.g., an animal inwhich expression of one or more of the subject ACE-2 proteins isdisrupted). Such transgenic animals can serve as animal models forstudying cellular and/or tissue disorders comprising mutated ormis-expressed ACE-2 alleles or for use in drug screening. Alternatively,such transgenic animals can be useful for expressing recombinant ACE-2polypeptides.

The invention further features assays and kits for determining whetheran individual's ACE-2 genes and/or proteins are defective or deficient(e.g in activity and/or level), and/or for determining the identity ofACE-2 alleles. In one embodiment, the method comprises the step ofdetermining the level of ACE-2 protein, the level ACE-2 mRNA and/or thetranscription rate of an ACE-2 gene. In another preferred embodiment,the method comprises detecting, in a tissue of the subject, the presenceor absence of a genetic alteration, which is characterized by at leastone of the following: a deletion of one or more nucleotides from a gene;an addition of one or more nucleotides to the gene; a substitution ofone or more nucleotides of the gene; a gross chromosomal rearrangementof the gene; an alteration in the level of a messenger RNA transcript ofthe gene; the presence of a non-wild type splicing pattern of amessenger RNA transcript of the gene; and/or a non-wild type level ofthe ACE-2 protein.

For example, detecting a genetic alteration or the presence of aspecific polymorphic region can include (i) providing a probe/primercomprised of an oligonucleotide which hybridizes to a sense or antisensesequence of an ACE-2 gene or naturally occurring mutants thereof, or 5′or 3′ flanking sequences naturally associated with the ACE-2 gene; (ii)contacting the probe/primer with an appropriate nucleic acid containingsample; and (iii) detecting, by hybridization of the probe/primer to thenucleic acid, the presence or absence of the genetic alteration.Particularly preferred embodiments comprise: 1) sequencing at least aportion of an ACE-2 gene, 2) performing a single strand conformationpolymorphism (SSCP) analysis to detect differences in electrophoreticmobility between mutant and wild-type nucleic acids; and 3) detecting orquantitating the level of an ACE-2 protein in an immunoassay using anantibody which is specifically immunoreactive with a wild-type ormutated ACE-2 protein.

Information obtained using the diagnostic assays described herein (aloneor in conjunction with information on another genetic defect, whichcontributes to the same disease) is useful for diagnosing or confirmingthat a symptomatic subject (e.g. a subject symptomatic for hypertension,hypotension, CHF, or a kinetensin-associated condition), has a geneticdefect (e.g. in an ACE-2 gene or in a gene that regulates the expressionof an ACE-2 gene), which causes or contributes to the particular diseaseor disorder. Alternatively, the information (alone or in conjunctionwith information on another genetic defect, which contributes to thesame disease) can be used prognostically for predicting whether anon-symptomatic subject is likely to develop a disease or condition,which is caused by or contributed to by an abnormal ACE-2 activity orprotein level (e.g. hypertension, hypotension, CHF, or akinetensin-associated condition) in a subject. In particular, the assayspermit to ascertain an individual's predilection to develop a conditionassociated with a mutation in ACE-2, where the mutation is a singlenucleotide polymorphism (SNP). Based on the prognostic information, adoctor can recommend a regimen (e.g. diet or exercise) or therapeuticprotocol useful for preventing or prolonging onset of the particulardisease or condition in the individual.

In addition, knowledge of the particular alteration or alterations,resulting in defective or deficient ACE-2 genes or proteins in anindividual, alone or in conjunction with information on other geneticdefects contributing to the same disease (the genetic profile of theparticular disease) allows customization of therapy for a particulardisease to the individual's genetic profile, the goal of“pharmacogenomics”. For example, an individual's ACE-2 genetic profileor the genetic profile of a disease or condition, to which ACE-2 geneticalterations cause or contribute, can enable a doctor: 1) to moreeffectively prescribe a drug that will address the molecular basis ofthe disease or condition; and 2) to better determine the appropriatedosage of a particular drug. For example, the expression level of ACE-2proteins, alone or in conjunction with the expression level of othergenes, known to contribute to the same disease, can be measured in manypatients at various stages of the disease to generate a transcriptionalor expression profile of the disease. Expression patterns of individualpatients can then be compared to the expression profile of the diseaseto determine the appropriate drug and dose to administer to the patient.

The ability to target populations expected to show the highest clinicalbenefit, based on the ACE-2 or disease genetic profile, can enable: 1)the repositioning of marketed drugs with disappointing market results;2) the rescue of drug candidates whose clinical development has beendiscontinued as a result of safety or efficacy limitations, which arepatient subgroup-specific; and 3) an accelerated and less costlydevelopment for drug candidates and more optimal drug labeling (e.g.since the use of ACE-2 as a marker is useful for optimizing effectivedose).

In another aspect, the invention provides methods for identifying acompound which modulates an ACE-2 activity, e.g. the interaction betweenan ACE-2 polypeptide and a target peptide, e.g., angiotensin I, a kinin,kinetensin or neurotensin. In a preferred embodiment, the methodincludes the steps of (a) forming a reaction mixture including: (i) anACE-2 polypeptide, (ii) an ACE-2 binding partner (e.g., a targetpeptide, such as angiotensin I or kinetensin), and (iii) a testcompound; and (b) detecting interaction of the ACE-2 polypeptide and theACE-2 binding protein. A statistically significant change (potentiationor inhibition) in the interaction of the ACE-2 polypeptide and ACE-2binding protein in the presence of the test compound, relative to theinteraction in the absence of the test compound, indicates a potentialagonist (mimetic or potentiator) or antagonist (inhibitor) of ACE-2bioactivity for the test compound. The reaction mixture can be acell-free protein preparation, e.g., a reconstituted protein mixture ora cell lysate, or it can be a recombinant cell including a heterologousnucleic acid recombinantly expressing the ACE-2 binding partner.

In preferred embodiments, the step of detecting interaction of the ACE-2and ACE-2 binding partner (e.g., angiotensin I or kinetensin) is acompetitive binding assay.

In preferred embodiments, at least one of the ACE-2 polypeptide and theACE-2 binding partner comprises a detectable label, and interaction ofthe ACE-2 and ACE-2 binding partner is quantified by detecting the labelin the complex. The detectable label can be, e.g., a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. In otherembodiments, the complex is detected by an immunoassay.

The invention also provides a methods for identifying an ACE-2therapeutic, comprising contacting in a reaction mixture an ACE-2polypeptide, a target peptide or analog thereof or portion thereof, anda test compound, in conditions wherein, but for the presence of the testcompound, the ACE-2 polypeptide cleaves one or more amino acids from thetarget peptide or analog thereof or portion thereof to produce an ACE-2target peptide conversion product, and detecting the presence of atleast one of the target peptide or analog thereof or portion thereof,the ACE-2 target peptide conversion product, and one or more aminoacids. A preferred method for determining the presence and/or the amountof at least one of the target peptide or analog thereof or portionthereof, the ACE-2 target peptide conversion product, and one or moreamino acids comprises obtaining a mass spectrum of the reaction mixtureor of a part thereof.

Yet another exemplary embodiment provides an assay for screening testcompounds to identify agents which modulate the amount of ACE-2 producedby a cell. In one embodiment, the screening assay comprises contacting acell transfected with a reporter gene operably linked to an ACE-2promoter with a test compound and determining the level of expression ofthe reporter gene. The reporter gene can encode, e.g., a gene productthat gives rise to a detectable signal such as: color, fluorescence,luminescence, cell viability, relief of a cell nutritional requirement,cell growth, and drug resistance. For example, the reporter gene canencode a gene product selected from the group consisting ofchloramphenicol acetyl transferase, luciferase, beta-galactosidase andalkaline phosphatase.

Also within the scope of the invention are methods for treating diseasesor disorders which are associated with an aberrant ACE-2 level oractivity or which can benefit from modulation of the activity or levelof ACE-2, in particular diseases or conditions which are improved bymodulation of the level of one or more angiotensin I conversionproducts, e.g., by an increase or decrease in the production ofAng.(1-9), Ang.(1-5), and/or Ang.(1-8) (angiotensin II); conditions thatare improved by modulation of kinetensin or kinetensin (1-8) level;conditions that are improved by modulation of bradykinin (1-8) orbradykinin (1-7); or conditions that are improved by modulation ofneurotensin (1-13) or neurotensin (1-12). Thus, the invention providesmethods for treating hypertension, CHF, inflammatory reactions, allergicreactions, and methods to reduce pain. The methods compriseadministering, e.g., either locally or systemically to a subject, apharmaceutically effective amount of a composition comprising an ACE-2therapeutic. Depending on the condition, the therapeutic can be an ACE-2agonist or an ACE-2 antagonist. For example, an ACE-2 antagonisttherapeutic can be administered to a subject having hypertension or CHF.In another embodiment, an ACE agonist is administered locally to asubject to reduce the inflammation and pain resulting from an insectsting or bite, which was accompanied by an injection of bradykinin.

In a particular embodiment of the invention, an ACE-2 antagonist isadministered to a subject alone or together with an ACE antagonist.Thus, a dual therapy comprising administering to a subject an antagonistof ACE-2 and an antagonist of ACE can be used to prevent theaccumulation of angiotensin II, e.g., to thereby reduce the bloodpressure of the subject and prevent the development or appearance ofconditions related thereto.

The invention also provides methods for identifying other potentialsubstrates of an ACE-2 polypeptide as well as the product of theenzymatic reaction. In a preferred embodiment, the method comprisescontacting a preparation containing an ACE-2 polypeptide with a testcompound, e.g., a peptide, for a time sufficient for the enzymaticreaction to occur, and subjecting the reaction mixture, or a portionthereof, to mass spectrometry. The comparison of the mass spectra of thetest compound with that of the reaction mixture after incubation toallow the enzymatic reaction to occur, will indicate whether the testcompound was converted into a new compound, in which case the testcompound is a substrate of the ACE-2 polypeptide.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1(A-B) shows the nucleotide sequence of a full length cDNA encodinghuman ACE-2 including 5′ and 3′ untranslated regions and codingsequences (SEQ ID NO:1) and the deduced amino acid sequence of the ACE-2protein (SEQ ID NO 2). The signal sequence is underlined, and the zincbinding domain (ZBD) and transmembrane (TMD) domain are boxed. Theposition of the introns is indicated. The two single chain polymorphismsare indicated by boxes around the nucleotides.

FIG. 2(A-D) shows an alignment of the amino acid sequence of human ACE-2having SEQ ID NO:2 with human testicular ACE (hu-ACET; SEQ ID NO:4;GenBank Accession No. P22966), murine testicular ACE (mu-ACET; SEQ IDNO:5; GenBank Accession No. P22967), rabbit testicular ACE (rb-ACET; SEQID NO:6; GenBank Accession No. P22968), human endothelial ACE hu-ACE;SEQ ID NO:7; GenBank Accession No. P12821; U.S. Pat. No. 5,539,045 bySoubrier et al.; and described in Soubrier et al. (1988) Proc. Natl.Acad. Sci. USA 85:9386), murine endothelial ACE (mu-ACE; SEQ ID NO:8;GenBank Accession No. P09470), rat endothelial ACE (rat-ACE; SEQ IDNO:9; GenBank Accession No. P47820) and rabbit endothelial ACE (rb-ACE;SEQ ID NO:10; GenBank Accession No. P12822). Stars indicate amino acidswhich are common to all sequences. Two dots indicate that all aminoacids at that position are conserved and one dot indicates that two ormore sequences share the amino acid at that position, but that at leastone sequence has an amino acid that is not a conservative substitutionat that position. The zinc binding domain (ZBD) and the transmembranedomain (TMD) are boxed.

FIG. 3(A-C) shows an amino acid alignment of the human ACE-2 proteinhaving SEQ ID NO:2 with human testicular ACE (HUM_tACE; SEQ ID NO:4;GenBank Accession No. P22966), human endothelial ACE (HUM_ACE; SEQ IDNO:7; GenBank Accession No. P12821, Drosophila melanogaster ACE(DROME_ACE; SEQ ID NO:11; GenBank Accession No. Q10714), and C. elegansACE (CE_ACE) SEQ ID NO:12; GenBank Accession No. U56966). Stars indicateamino acids which are common to all sequences. Two dots indicate thatall amino acids at that position are conserved and one dot indicatesthat two or more sequences share the amino acid at that position, butthat at least one sequence has an amino acid that is not a conservativesubstitution at that position. The zinc binding domain (ZBD) and thetransmembrane domain (TMD) are boxed.

FIG. 4 shows the activity of baculovirus expressed ACE-2 in differentfractions from the column.

FIG. 5 (panels A and B) show mass spectra of angiotensin I (panel A) andits conversion product after reaction with ACE-2 (panel B).

FIG. 6 (panels A and B) show mass spectrum of neurotensin (1-13) (panelA) and its conversion product after reaction with ACE-2 (panel B).

FIG. 7 (panels A and B) show mass spectrum of bradykinin (panel A) andits conversion product after reaction with ACE-2 (panel B).

FIG. 8 shows at least some angiotensin I conversion products and enzymescatalyzing these reactions (E=endopeptidase, e.g., neprilysin;AP=aminopeptidase).

FIG. 9A is a diagram of the structure of the human ACE-2 gene and thelocation of the polymorphisms.

FIG. 9B is a diagram of the cDNA sequence of human ACE-2 (SEQ ID NO:1)indicating the position of the introns and indicating the polymorphisms.

FIG. 10 panels A and B show ACE-2 target peptides.

FIGS. 11A-C depict mouse surface EKGs in wild type (WT) and ACE-2transgenic mice at 3 days (FIG. 11A), 14 days (FIG. 11B) and 28 days(FIG. 11C).

FIG. 12 depicts the results from the Holter monitoring experimentsshowing evidence of non sustained ventricular tachycardia whichprogressed into ventricular fibrillation and ultimately resulted inasystole and death in one of the transgenic mice that were tested.

FIG. 13A is a diagram of the structure of the human ACE-2 gene and thelocation of the polymorphisms in the Caucasian as well as the Asianpopulation.

FIG. 13B is a diagram of the cDNA sequence of human ACE-2 (SEQ ID NO:1)indicating the position of the introns and indicating the polymorphismsin the Caucasian as well as the Asian population.

DETAILED DESCRIPTION OF THE INVENTION

1. General

The invention is based at least in part on the discovery of a geneencoding a protein having regions which are significantly homologous toregions of known angiotensin converting enzymes (ACEs). Thus, the genesand proteins disclosed herein are referred to as Angiotensin ConvertingEnzyme 2 (ACE-2) genes and proteins. The sequence of the full lengthcDNA encoding ACE-2 was determined from a clone obtained from a cDNAlibrary prepared from mRNA of a human heart of a subject who hadcongestive heart failure. The cDNA encoding the full length human ACE-2protein and comprising 5′ and 3′ untranslated regions is 3396nucleotides long and has the nucleotide sequence shown in FIG. 1 and isset forth as SEQ ID NO:1. The full length human ACE-2 protein is 805amino acids long and has the amino acid sequence shown in FIG. 1 and setforth in SEQ ID NO:2. The coding portion (open reading frame) of SEQ IDNO:1 is set forth as SEQ ID NO:3 and corresponds to nucleotides 82 to2496 of SEQ ID NO:1. The cDNA encoding the full length ACE-2 protein hasbeen deposited at the American Type Culture Collection (12301 ParklawnDrive, Rockville, Md.) on Dec. 3, 1997 has been assigned ATCCDesignation No. 209510.

The protein comprises a signal peptide from amino acid 1 to amino acid18, which is encoded by nucleotides 82 to 135 of SEQ ID NO:1. Thus, themature ACE-2 protein has 787 amino acids and has the amino acid sequencefrom amino acid 19 to amino acid 805 of SEQ ID NO:2.

ACE-2 protein further comprises several functional domains. ACE-2comprises a zinc binding domain (ZBD) from amino acid 374 to amino acid378 of SEQ ID NO:2, which is encoded by the nucleotide sequence fromnucleotide 1201 to 1215 of SEQ ID NO:1 and referred to herein as minimumzinc binding domain. It is in fact likely that at least some of theadjacent amino acids participate in binding zinc. This minimum zincbinding domain has the amino acid sequence HHEMGH (SEQ ID NO:14), and isidentical to the zinc binding domain that is present in all ACE proteins(see below) which have been identified as being located in the catalyticsite of the enzyme (Lattion et al. (1989) FEBS Letters 252:99). Sinceamino acids 372-381 of SEQ ID NO:2 are conserved in all ACE proteins(see below), it is likely that amino acids 372, 373, 379, 380, and 381of SEQ ID NO:2 are involved in binding zinc. In addition, all the aminoacids which have been reported as interacting with the zinc atom orinvolved in catalysis in ACE proteins are present in ACE-2. Thus, bycomparison, His 374, 378 and Glu 402 are probably the amino acidscoordinating the zinc atom and Glu 375 and His 417 are probably involvedin catalysis. It is also believed that Glu 406 is involved in thecatalytic activity of the enzyme.

ACE-2 also has a hydrophobic region in its C-terminal region, having theamino acid sequence from about amino acid 741 to about amino acid 765 ofSEQ ID NO:2 and is encoded by the nucleotide sequence from aboutnucleotide 2302 to about nucleotide 2376 of SEQ ID NO:1. Thishydrophobic region is a transmembrane domain, similar to that present inACE proteins (see below).

A BLAST search (Altschul et al. (1990) J. Mol. Biol. 215:403) of thenucleic acid and the amino acid sequences of ACE-2 revealed that certainportions of the ACE-2 protein and cDNA have a significant homology tocertain regions of previously identified angiotensin converting enzymes.Two forms of ACE proteins have been described previously: a larger form,referred to as endothelial or somatic ACE, since it is present innumerous somatic tissues, including vascular endothelium, renal tubularepithelium, ciliated gut epithelium, stimulated macrophages, areas ofthe brain and testis. The smaller form of ACE is referred to as thetesticular form, since it is found essentially only in developing spermcells in the testis.

The previously cloned mature endothelial human ACE protein consists of1277 amino acid residues and is organized into two large homologousdomains, each bearing a putative active site (Soubrier et al. (1988)Proc. Natl. Acad. Sci. U.S.A. 85:9386). Each of these two domainscontain short amino acid sequences identical to those located aroundcritical residues of the active site of other metalloproteinases(theimmolysin, neutral endopeptidase, and collagenase) and thereforebears a putative active site. Zinc has been reported as essential forthe activity of ACE (Bunning and Riordan (1985) J. Inorg. Biochem.24:183). Only one of these sites is probably involved in catalyzingangiotensin II, since only one Zn atom has been reported to beassociated per ACE molecule. However, another study showed that bothdomains have activity, as shown by measuring the activity of each domainas a separate protein. The human ACE protein exist in a soluble and in amembrane bound form. Membrane attachment is likely to be mediated by theC-terminal hydrophobic sequence located near the carboxyterminus of theprotein (Soubrier et al, supra), which is also present in ACE-2. Thehuman testicular ACE contains 732 residues (including the signalpeptide) contains only one of these two large repetitive domains ofendothelial ACE, i.e., the carboxyterminal domain (WO 91/00354 andEhlers et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86:7741). This domainis located N-terminal of a 67 amino acids stretch and a serine-threoninerich region that is specific to the testicular form of ACE. TesticularACE is encoded by the same gene as that encoding the larger ACE protein,but is encoded by a mRNA transcribed from a site located in the 12^(th)intron of the ACE gene encoding the endothelial protein (Howard et al.(1990) Mol. Cell. Biol. 10:4294).

As shown in FIGS. 2 and 3, which show amino acid sequence alignments ofACE-2 with human, mouse, rabbit, rat, testicular and endothelial ACEproteins and Drosophila and C. elegans ACE proteins, certain portions ofACE-2 are homologous to certain regions of ACE proteins. In particular,the zinc binding domain is conserved in all ACE proteins. Thus, sincethe zinc binding domain is located in the catalytic site of the proteinthat is responsible for its peptidase activity, the function of ACE-2 islikely to be similar to that of other ACE proteins. In fact, aspredicted by the homology between the amino acid sequences of ACE-2 andACE, ACE-2 is capable of hydrolyzing angiotensin I and thereby cleavingoff the last C-terminal amino acid (i.e., leucine) from angiotensin I.This 9 amino acid peptide (“Ang.(1-9)”) can be further hydrolyzed by ACEinto a 5 amino acid peptide containing the first five amino acids fromangiotensin I (see FIG. 8). As shown in the Examples, ACE-2 is alsocapable of catalyzing the hydrolysis of other peptides, includingkinins, neurotensin and kinetensin, and is involved in regulating bloodpressure in a similar manner as endothelial ACE protein.

Another homology between ACE-2 and other ACE proteins is the presence ofa transmembrane domain in the carboxy terminal portion of the proteins.Thus, ACE-2 can be in a membrane bound form. ACE proteins have also beenfound in a soluble form, which may result either from leakage of theprotein from the surface or, from specific hydrolysis by a protease, orthe soluble form may be encoded by a differentially spliced mRNA.Accordingly, ACE-2 exists in a soluble form.

The amino acid sequence alignment indicates the existence of otherregions of strong homology between ACE-2 and ACE proteins (see FIGS. 2and 3). However, the overall similarity of ACE-2 with ACE proteins isrelatively weak. In fact, the overall percent identity and similaritybetween human ACE-2 and the human testicular ACE protein (which is theACE protein with which ACE-2 has the highest overall similarity) isabout 42.9% and 62% respectively. At the nucleotide level, human ACE-2and human testicular ACE have about 50.8% identity.

Northern blot hybridizations indicated that the mRNA encoding humanACE-2 is about 4 kb, which correlates with the size of the full lengthcDNA. ACE-2 mRNA is expressed predominantly in kidney, heart, andtestis. Thus, the pattern of expression of ACE-2 is more specific thanthat of endothelial ACE.

Accordingly, the invention provides nucleic acids encoding ACE-2proteins, fragments thereof and homologs or variants thereof. Theinvention also provides ACE-2 polypeptides, fragments thereof andhomologs or variants thereof.

Based at least on the observation of sequence homologies between ACE-2and angiotensin converting enzymes, as well as the fact that ACE-2 iscapable of hydrolyzing angiotensin I into Ang.(1-9), the inventionfurther provides methods and compositions for regulating arterial bloodpressure, which can be used, e.g., for treating or preventing arterialhypertension, hypotension, or congestive heart failure. In addition,since ACEs have been shown to hydrolyze other peptides, e.g., kinins,such as bradykinin, the compositions of the invention can also be usedas analgesics, or for treating inflammatory diseases or conditions.Based at least on the fact that ACE-2 is also homologous to thetesticular ACE, methods and compositions of the invention could also beused to treat and prevent diseases or conditions relating to fertility.Furthermore, based on the observation, described herein, that ACE-2catalyzes the hydrolysis of kinetensin, ACE-2 therapeutics can be usedfor treating and preventing diseases associated with excessive histaminerelease or abnormal blood vessel permeability. Moreover, based on theobservation described herein that ACE-2 transgenic mice exhibit nonsustained ventricular tachycardia which progresses into ventricularfibrillation and ultimately results in asystole and death, methods andcompositions of the invention may be used to treat disorders associatedwith abnormalities in the conduction system of the heart, such asarrhythmias. An arrhythmia is an abnormality or irregularity in theheart rhythm. An arrhythmia results when there is a disturbance in theconduction system of the heart, for example, due to faulty production ofelectrical impulses or faulty conduction of impulses as they passthrough the system. Examples of arrhythmias include heart block (e.g,atrioventricular heart block), atrial flutter, atrial fibrillation,ventricular fibrillation, and extrasystole (premature contraction).

The invention further provides diagnostic and prognostic methods, e.g.,methods for determining whether a subject is at risk of developing orhas developed a disease associated with an aberrant ACE-2 activity,e.g., arterial hypertension, hypotension, arrhythmia, or CHF. Suchassays can, for example, consist of determining whether the subject hasa genetic alteration in an ACE-2 gene or an abnormal level of ACE-2protein. Also within the scope of the invention are methods foridentifying ACE-2 therapeutics, i.e., compounds, which are either ACE-2agonists or ACE-2 antagonists.

Other disorders which may be treated or diagnosed by the methodsdescribed herein include, but are not limited to, azotemia, renaldisease, renal failure, glomerular disease, glomerulonephritis(vasculitis), nephritis, acute tubular necrosis, proteinuria, hematuria,pyuria, pyelonephritis, polyuria, fluid and electrolyte (e.g., sodiumand patassium) disturbances, hypovolemia, hyponatremia, hypematremia,hypokalemia (Liddle's Syndrome, Bartter's Syndrome), hyperkalemia(Gordon's Syndrome), acidosis, alkalosis and hyperchloremic metabolicdisorders.

Other aspects of the invention are described below or will be apparentto those skilled in the art in light of the present disclosure.

2. Definitions

For convenience, the meaning of certain terms and phrases employed inthe specification, examples, and appended claims are provided below.

The term “ACE-2 nucleic acid” refers to a nucleic acid encoding an ACE-2protein, such as nucleic acids having SEQ ID NO:1 or 3, fragmentsthereof, complement thereof, and derivatives thereof.

The terms “ACE-2 polypeptide” and “ACE-2 protein” are intended toencompass polypeptides comprising the amino acid sequence SEQ ID NO:2,fragments thereof, and homologs thereof and include agonist andantagonist polypeptides.

The term “ACE-2 therapeutic” refers to various forms of ACE-2polypeptides, as well as peptidomimetics, nucleic acids, or smallmolecules, which can modulate at least one activity of an ACE-2polypeptide, e.g., interaction with and/or hydrolysis of a targetpeptide, by mimicking or potentiating (agonizing) or inhibiting(antagonizing) the effects of a naturally-occurring ACE-2 polypeptide.An ACE-2 therapeutic which mimics or potentiates the activity of awild-type ACE-2 polypeptide is a “ACE-2 agonist”. Conversely, an ACE-2therapeutic which inhibits the activity of a wild-type ACE-2 polypeptideis a “ACE-2 antagonist”.

The term “agonist”, as used herein, is meant to refer to an agent thatmimics or upregulates (e.g. potentiates or supplements) an ACE-2bioactivity. An ACE-2 agonist can be a wild-type ACE-2 protein orderivative thereof having at least one bioactivity of the wild-typeACE-2. An ACE-2 therapeutic can also be a compound that upregulatesexpression of an ACE-2 gene or which increases at least one bioactivityof an ACE-2 protein. An agonist can also be a compound which increasesthe interaction of an ACE-2 polypeptide with another molecule, e.g, atarget peptide.

“Antagonist” as used herein is meant to refer to an agent thatdownregulates (e.g. suppresses or inhibits) at least one ACE-2bioactivity. An ACE-2 antagonist can be a compound which inhibits ordecreases the interaction between an ACE-2 protein and another molecule,e.g., a target peptide, such as angiotensin I or a kinin. Accordingly, apreferred antagonist is a compound which inhibits or decreaseshydrolysis of a target peptide. An antagonist can also be a compoundthat downregulates expression of an ACE-2 gene or which reduces theamount of ACE-2 protein present. An ACE-2 antagonist can be a dominantnegative form of an ACE-2 polypeptide, e.g., a form of an ACE-2polypeptide which is capable of interacting with a target peptide, e.g.,angiotensin I, but which is not capable of hydrolysing the targetpeptide. The ACE-2 antagonist can also be a nucleic acid encoding adominant negative form of an ACE-2 polypeptide, an ACE-2 antisensenucleic acid, or a ribozyme capable of interacting specifically with anACE-2 RNA. Yet other ACE-2 antagonists are molecules which bind to anACE-2 polypeptide and inhibit its action. Such molecules includepeptides, e.g., forms of ACE-2 target peptides which do not havebiological activity, and which inhibit hydrolysis of target peptides byACE-2 by competition with the target peptides. Thus, such peptides willbind the active site of ACE-2 and prevent it from interacting withtarget peptides, e.g., angiotensin I. Yet other ACE-2 antagonistsinclude antibodies interacting specifically with an epitope of an ACE-2molecule, such that binding interferes with hydrolysis. In yet anotherpreferred embodiment, the ACE-2 antagonist is a small molecule, such asa molecule capable of inhibiting the interaction between an ACE-2polypeptide and a target peptide and/or binding to the catalytic site ofthe enzyme. Alternatively, the small molecule can be antagonist byinteracting with sites other than the catalytic site, and inhibit thecatalytic activity of ACE-2 by, e.g., altering the tertiary orquaternary structure of the enzyme.

The term “ACE-2 substrate conversion product” or “ACE-2 target peptideconversion product” refers to a product, in particular a peptide, whichresult from the enzymatic cleavage of the ACE-2 substrate by ACE-2.

The term “kinetensin conversion product” refers to a product resultingfrom enzymatic cleavage of kinetensin by ACE-2, e.g., kinetensin (1-8)having SEQ ID NO:24.

The term “angiotensin conversion product” refers to a peptide resultingfrom hydrolysis of angiotensin I. Examples of such peptides and theenzymes catalyzing their production are shown in FIG. 7. Angiotensinconversion products include Ang.(1-9), Ang.(1-8), Ang.(1-7), Ang.(1-6),Ang.(1-5), Ang. III, and (des-Asp)AngI.

The term “angiotensin (1-9) or “Ang.(1-9)” refers to an angiotensin Ipeptide (DRVYIHPFHL; SEQ ID NO:15) in which the C-terminal leucine isabsent, i.e., a peptide having the amino acid sequence DRVYIHPFH (SEQ IDNO:16). The term “angiotensin (1-8) or “Ang.(1-8)” refers to angiotensinII, i.e., a peptide having the amino acid sequence DRVYIHPF (SEQ IDNO:17). The term “angiotensin (1-7)” or “Ang.(1-7)” refers to anangiotensin I peptide in which the last 3 C-terminal amino acids areabsent, i.e., a peptide having the amino acid sequence DRVYIHP (SEQ IDNO:18). The term “angiotensin (1-6) or “Ang.(1-6)” refers to anangiotensin I peptide in which the last 4 C-terminal amino acids areabsent, i.e., a peptide having the amino acid sequence DRVYIH (SEQ IDNO:19). The term “angiotensin (1-5)” or “Ang.(1-5)” refers to anangiotensin I peptide in which the last 5 C-terminal amino acids areabsent, i.e., a peptide having the amino acid sequence DRVYI (SEQ IDNO:20). The term “(des-Asp)Ang. I” refers to an angiotensin I peptidelacking the N-terminal amino acid, i.e., a peptide having the amino acidsequence RVYIHPFHL (SEQ ID NO:21). The term “angiotensin III” or “Ang.III” refers to an angiotensin I peptide lacking the C-terminal aminoacid and the last two N-terminal amino acids, i.e., a peptide having theamino acid sequence RVYIHPF (SEQ ID NO:22).

The term “allele”, which is used interchangeably herein with “allelicvariant” refers to alternative forms of a gene or portions thereof.Alleles occupy the same locus or position on homologous chromosomes.When a subject has two identical alleles of a gene, the subject is saidto be homozygous for the gene or allele. When a subject has twodifferent alleles of a gene, the subject is said to be heterozygous forthe gene. Alleles of a specific gene can differ from each other in asingle nucleotide, or several nucleotides, and can includesubstitutions, deletions, and insertions of nucleotides. An allele of agene can also be a form of a gene containing a mutation.

The term “allelic variant of a polymorphic region of an ACE-2 gene”refers to a region of an ACE-2 gene having one of several nucleotidesequences found in that region of the gene in other individuals.

The term “analog of a target peptide of an ACE-2 polypeptide” refers toa compound which has sufficient structural similarities with the targetpeptide, that it shares a biological activity of the target peptide,e.g., binding of the target peptide to an ACE-2 polypeptide or theability to be hydrolyzed by an ACE-2 polypeptide. An analog can be, e.g,a peptidomimetic or any modified peptide.

“Biological activity” or “bioactivity” or “activity” or “biologicalfunction”, which are used interchangeably, for the purposes herein meansan effector or antigenic function that is directly or indirectlyperformed by an ACE-2 polypeptide (whether in its native or denaturedconformation), or by any subsequence thereof. Biological activitiesinclude binding to a target peptide, e.g., angiotensin I, a kinin, orkinetensin; binding to other proteins or molecules; the capability tocatalyze hydrolysis of a target peptide (function as a carboxypeptidaseor endopeptidase), e.g., angiotensin I, a kinin, or kinetensin; andinteraction with a metal ion, e.g., Zn²⁺ Co²⁺, and Mn²⁺. An ACE-2bioactivity can be modulated by affecting directly an ACE-2 polypeptide.Alternatively, an ACE-2 bioactivity can be modulated by modulating thelevel of an ACE-2 polypeptide, such as by modulating expression of anACE-2 gene.

As used herein the term “bioactive fragment of an ACE-2 polypeptide”refers to a fragment of a full-length ACE-2 polypeptide, wherein thefragment specifically mimics or antagonizes the activity of a wild-typeACE-2 polypeptide. The bioactive fragment preferably is a fragmentcapable of interacting with a target peptide, such as angiotensin I orkinetensin.

The term “an aberrant activity”, as applied to an activity of apolypeptide such as ACE-2, refers to an activity which differs from theactivity of the wild-type or native polypeptide or which differs fromthe activity of the polypeptide in a healthy subject. An activity of apolypeptide can be aberrant because it is stronger than the activity ofits native counterpart. Alternatively, an activity can be aberrantbecause it is weaker or absent relative to the activity of its nativecounterpart. An aberrant activity can also be a change in an activity.For example an aberrant polypeptide can interact with a different targetpeptide. A cell can have an aberrant ACE-2 activity due tooverexpression or underexpression of the gene encoding ACE-2.

The term “abnormal blood pressure” refers to hypertension andhypotension.

“Cells,” “host cells” or “recombinant host cells” are terms usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A “chimeric polypeptide” or “fusion polypeptide” is a fusion of a firstamino acid sequence encoding one of the subject ACE-2 polypeptides witha second amino acid sequence defining a domain (e.g. polypeptideportion) foreign to and not substantially homologous with any domain ofan ACE-2 polypeptide. A chimeric polypeptide may present a foreigndomain which is found (albeit in a different polypeptide) in an organismwhich also expresses the first polypeptide, or it may be an“interspecies”, “intergenic”, etc. fusion of polypeptide structuresexpressed by different kinds of organisms. In general, a fusionpolypeptide can be represented by the general formula X-ACE-2-Y, whereinACE-2 represents a portion of the polypeptide which is derived from anACE-2 polypeptide, and X and Y are independently absent or representamino acid sequences which are not related to an ACE-2 sequence in anorganism, including naturally occurring mutants.

The term “nucleotide sequence complementary to the nucleotide sequenceset forth in SEQ ID NO: x” refers to the nucleotide sequence of thecomplementary strand of a nucleic acid strand having SEQ ID NO: x. Theterm “complementary strand” is used herein interchangeably with the term“complement”. The complement of a nucleic acid strand can be thecomplement of a coding strand or the complement of a non-coding strand.When referring to double stranded nucleic acids, the complement of anucleic acid having SEQ ID NO: x refers to the complementary strand ofthe strand having SEQ ID NO: x or to any nucleic acid having thenucleotide sequence of the complementary strand of SEQ ID NO: x. Whenreferring to a single stranded nucleic acid having the nucleotidesequence SEQ ID NO: x, the complement of this nucleic acid is a nucleicacid having a nucleotide sequence which is complementary to that of SEQID NO: x. The nucleotide sequences and complementary sequences thereofare always given in the 5′ to 3′ direction.

A “delivery complex” shall mean a targeting means (e.g. a molecule thatresults in higher affinity binding of a gene, protein, polypeptide orpeptide to a target cell surface and/or increased cellular or nuclearuptake by a target cell). Examples of targeting means include: sterols(e.g. cholesterol), lipids (e.g. a cationic lipid, virosome orliposome), viruses (e.g. adenovirus, adeno-associated virus, andretrovirus) or target cell specific binding agents (e.g. ligandsrecognized by target cell specific receptors). Preferred complexes aresufficiently stable in vivo to prevent significant uncoupling prior tointernalization by the target cell. However, the complex is cleavableunder appropriate conditions within the cell so that the gene, protein,polypeptide or peptide is released in a functional form.

As is well known, genes may exist in single or multiple copies withinthe genome of an individual. Such duplicate genes may be identical ormay have certain modifications, including nucleotide substitutions,additions or deletions, which all still code for polypeptides havingsubstantially the same activity. The term “DNA sequence encoding anACE-2 polypeptide” may thus refer to one or more genes within aparticular individual. Moreover, certain differences in nucleotidesequences may exist between individual organisms, which are calledalleles. Such allelic differences may or may not result in differencesin amino acid sequence of the encoded polypeptide yet still encode apolypeptide with the same biological activity.

A disease, disorder or condition “associated with” or “characterized by”an aberrant ACE-2 activity refers to a disease, disorder or condition ina subject which is caused by or contributed to by an aberrant ACE-2activity.

“Histamine-associated condition” refers to a condition, disease ordisorder caused by or contributed to by excessive release of histamine,e.g., conditions resulting from excessive endothelial cell contraction,leakage of plasma into the tissues, and/or vasodilation. Examples ofsuch conditions include topical and systemic allergies, excema, asthma,and anaphylactic shock

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two peptides or between two nucleic acid molecules. Homology canbe determined by comparing a position in each sequence which may bealigned for purposes of comparison. When a position in the comparedsequence is occupied by the same base or amino acid, then the moleculesare identical at that position. A degree of homology or similarity oridentity between nucleic acid sequences is a function of the number ofidentical or matching nucleotides at positions shared by the nucleicacid sequences. A degree of identity of amino acid sequences is afunction of the number of identical amino acids at positions shared bythe amino acid sequences. A degree of homology or similarity of aminoacid sequences is a function of the number of amino acids, i.e.structurally related, at positions shared by the amino acid sequences.An “unrelated” or “non-homologous” sequence shares less than 40%identity, though preferably less than 25% identity, with one of theACE-2 sequences of the present invention.

The term “interact” as used herein is meant to include detectablerelationships or association (e.g. biochemical interactions) betweenmolecules, such as interaction between protein-protein, protein-nucleicacid, nucleic acid-nucleic acid, and protein-small molecule or nucleicacid-small molecule in nature.

The term “isolated” as used herein with respect to nucleic acids, suchas DNA or RNA, refers to molecules separated from other DNAs, or RNAs,respectively, that are present in the natural source of themacromolecule. For example, an isolated nucleic acid encoding one of thesubject ACE-2 polypeptides preferably includes no more than 10 kilobases(kb) of nucleic acid sequence which naturally immediately flanks theACE-2 gene in genomic DNA, more preferably no more than 5 kb of suchnaturally occurring flanking sequences, and most preferably less than1.5 kb of such naturally occurring flanking sequence. The term isolatedas used herein also refers to a nucleic acid or peptide that issubstantially free of cellular material, viral material, or culturemedium when produced by recombinant DNA techniques, or chemicalprecursors or other chemicals when chemically synthesized. Moreover, an“isolated nucleic acid” is meant to include nucleic acid fragments whichare not naturally occurring as fragments and would not be found in thenatural state. The term “isolated” is also used herein to refer topolypeptides which are isolated from other cellular proteins and ismeant to encompass both purified and recombinant polypeptides.

“Kinetensin (1-8)” refers to kinetensin (SEQ ID NO:23) lacking theC-terminal leucine, i.e., IARRHPYF (SEQ ID NO:24).

The term “kinetensin associated condition” refers to a conditionresulting from an abnormal level of kinetensin or kinetensin conversionproduct in plasma or in tissues of a subject and includes histamineassociated conditions.

The term “modulation” as used herein refers to both upregulation (i.e.,activation or stimulation (e.g., by agonizing or potentiating)) anddownregulation (i.e. inhibition or suppression (e.g., by antagonizing,decreasing or inhibiting)).

The term “mutated gene” refers to an allelic form of a gene, which iscapable of altering the phenotype of a subject having the mutated generelative to a subject which does not have the mutated gene. If a subjectmust be homozygous for this mutation to have an altered phenotype, themutation is said to be recessive. If one copy of the mutated gene issufficient to alter the genotype of the subject, the mutation is said tobe dominant. If a subject has one copy of the mutated gene and has aphenotype that is intermediate between that of a homozygous and that ofa heterozygous subject (for that gene), the mutation is said to beco-dominant.

The “non-human animals” of the invention include mammalians such asrodents, non-human primates, sheep, dog, cow, chickens, amphibians,reptiles, etc. Preferred non-human animals are selected from the rodentfamily including rat and mouse, most preferably mouse, though transgenicamphibians, such as members of the Xenopus genus, and transgenicchickens can also provide important tools for understanding andidentifying agents which can affect, for example, embryogenesis andtissue formation. The term “chimeric animal” is used herein to refer toanimals in which the recombinant gene is found, or in which therecombinant gene is expressed in some but not all cells of the animal.The term “tissue-specific chimeric animal” indicates that one of therecombinant ACE-2 genes is present and/or expressed or disrupted in sometissues but not others.

As used herein, the term “nucleic acid” refers to polynucleotides suchas deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA). The term should also be understood to include, as equivalents,analogs of either RNA or DNA made from nucleotide analogs, and, asapplicable to the embodiment being described, single (sense orantisense) and double-stranded polynucleotides.

The term “polymorphism” refers to the coexistence of more than one formof a gene or portion (e.g., allelic variant) thereof. A portion of agene of which there are at least two different forms, i.e., twodifferent nucleotide sequences, is referred to as a “polymorphic regionof a gene”. A polymorphic region can be a single nucleotide, theidentity of which differs in different alleles. A polymorphic region canalso be several nucleotides long.

A “polymorphic gene” refers to a gene having at least one polymorphicregion.

As used herein, the term “promoter” means a DNA sequence that regulatesexpression of a selected DNA sequence operably linked to the promoter,and which effects expression of the selected DNA sequence in cells. Theterm encompasses “tissue specific” promoters, i.e. promoters, whicheffect expression of the selected DNA sequence only in specific cells(e.g. cells of a specific tissue). The term also covers so-called“leaky” promoters, which regulate expression of a selected DNA primarilyin one tissue, but cause expression in other tissues as well. The termalso encompasses non-tissue specific promoters and promoters thatconstitutively express or that are inducible (i.e. expression levels canbe controlled).

The terms “protein”, “polypeptide” and “peptide” are usedinterchangeably herein when referring to a gene product.

The term “recombinant protein” refers to a polypeptide of the presentinvention which is produced by recombinant DNA techniques, whereingenerally, DNA encoding an ACE-2 polypeptide is inserted into a suitableexpression vector which is in turn used to transform a host cell toproduce the heterologous protein. Moreover, the phrase “derived from”,with respect to a recombinant ACE-2 gene, is meant to include within themeaning of “recombinant protein” those proteins having an amino acidsequence of a native ACE-2 polypeptide, or an amino acid sequencesimilar thereto which is generated by mutations including substitutionsand deletions (including truncation) of a naturally occurring form ofthe polypeptide.

“Small molecule” as used herein, is meant to refer to a composition,which has a molecular weight of less than about 5 kD and most preferablyless than about 4 kD. Small molecules can be nucleic acids, peptides,polypeptides, peptidomimetics, carbohydrates, lipids or other organic(carbon containing) or inorganic molecules. Many pharmaceuticalcompanies have extensive libraries of chemical and/or biologicalmixtures, often fungal, bacterial, or algal extracts, which can bescreened with any of the assays of the invention to identify compoundsthat modulate an ACE-2 bioactivity.

As used herein, the term “specifically hybridizes” or “specificallydetects” refers to the ability of a nucleic acid molecule of theinvention to hybridize to at least approximately 6, 12, 20, 30, 50, 100,150, 200, 300, 350, 400 or 425 consecutive nucleotides of a vertebrate,preferably an ACE-2 gene.

The term “target peptide” refers to a peptide which can be hydrolyzed byan ACE or ACE-2 protein. Target peptides include angiotensin I, kininssuch as bradykinin, kinetensin, enkephalins, and neuropeptides such assubstance P.

“Transcriptional regulatory sequence” is a generic term used throughoutthe specification to refer to DNA sequences, such as initiation signals,enhancers, and promoters, which induce or control transcription ofprotein coding sequences with which they are operably linked. Inpreferred embodiments, transcription of one of the ACE-2 genes is underthe control of a promoter sequence (or other transcriptional regulatorysequence) which controls the expression of the recombinant gene in acell-type in which expression is intended. It will also be understoodthat the recombinant gene can be under the control of transcriptionalregulatory sequences which are the same or which are different fromthose sequences which control transcription of the naturally-occurringforms of ACE-2 polypeptide.

As used herein, the term “transfection” means the introduction of anucleic acid, e.g., via an expression vector, into a recipient cell bynucleic acid-mediated gene transfer. “Transformation”, as used herein,refers to a process in which a cell's genotype is changed as a result ofthe cellular uptake of exogenous DNA or RNA, and, for example, thetransformed cell expresses a recombinant form of an ACE-2 polypeptideor, in the case of anti-sense expression from the transferred gene, theexpression of a naturally-occurring form of the ACE-2 polypeptide isdisrupted.

As used herein, the term “transgene” means a nucleic acid sequence(encoding, e.g., one of the ACE-2 polypeptides, or an antisensetranscript thereto) which has been introduced into a cell. A transgenecould be partly or entirely heterologous, i.e., foreign, to thetransgenic animal or cell into which it is introduced, or, is homologousto an endogenous gene of the transgenic animal or cell into which it isintroduced, but which is designed to be inserted, or is inserted, intothe animal's genome in such a way as to alter the genome of the cellinto which it is inserted (e.g., it is inserted at a location whichdiffers from that of the natural gene or its insertion results in aknockout). A transgene can also be present in a cell in the form of anepisome. A transgene can include one or more transcriptional regulatorysequences and any other nucleic acid, such as introns, that may benecessary for optimal expression of a selected nucleic acid.

A “transgenic animal” refers to any animal, preferably a non-humanmammal, bird or an amphibian, in which one or more of the cells of theanimal contain heterologous nucleic acid introduced by way of humanintervention, such as by transgenic techniques well known in the art.The nucleic acid is introduced into the cell, directly or indirectly byintroduction into a precursor of the cell, by way of deliberate geneticmanipulation, such as by microinjection or by infection with arecombinant virus. The term genetic manipulation does not includeclassical cross-breeding, or in vitro fertilization, but rather isdirected to the introduction of a recombinant DNA molecule. Thismolecule may be integrated within a chromosome, or it may beextrachromosomally replicating DNA. In the typical transgenic animalsdescribed herein, the transgene causes cells to express a recombinantform of one of the ACE-2 polypeptide, e.g. either agonistic orantagonistic forms. However, transgenic animals in which the recombinantACE-2 gene is silent are also contemplated, as for example, the FLP orCRE recombinase dependent constructs described below. Moreover,“transgenic animal” also includes those recombinant animals in whichgene disruption of one or more ACE-2 genes is caused by humanintervention, including both recombination and antisense techniques.

The term “treating” as used herein is intended to encompass curing aswell as ameliorating at least one symptom of the condition or disease.

The term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof preferred vector is an episome, i.e., a nucleic acid capable ofextra-chromosomal replication. Preferred vectors are those capable ofautonomous replication and/or expression of nucleic acids to which theyare linked. Vectors capable of directing the expression of genes towhich they are operatively linked are referred to herein as “expressionvectors”. In general, expression vectors of utility in recombinant DNAtechniques are often in the form of “plasmids” which refer generally tocircular double stranded DNA loops which, in their vector form are notbound to the chromosome. In the present specification, “plasmid” and“vector” are used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors which serve equivalent functions andwhich become known in the art subsequently hereto.

The term “wild-type allele” refers to an allele of a gene which, whenpresent in two copies in a subject results in a wild-type phenotype.There can be several different wild-type alleles of a specific gene,since certain nucleotide changes in a gene may not affect the phenotypeof a subject having two copies of the gene with the nucleotide changes.

3. Nucleic Acids of the Present Invention

The invention provides ACE-2 nucleic acids, homologs thereof, andportions thereof. Preferred nucleic acids have a sequence at least 70%,and more preferably 75% homologous and more preferably 80% and even morepreferably at least 85% homologous with a nucleotide sequence of anACE-2 gene, e.g., such as a sequence shown in one of SEQ ID NOs: 1 or 3or complement thereof or the ACE-2 nucleic acid having ATCC DesignationNo. 209510. Nucleic acids at least 90%, more preferably 95%, and mostpreferably at least about 98-99% homologous with a nucleic sequencerepresented in one of SEQ ID NOs: 1 or 3 or complement thereof are ofcourse also within the scope of the invention. In preferred embodiments,the nucleic acid is mammalian and in particularly preferred embodiments,includes all or a portion of the nucleotide sequence corresponding tothe coding region of one of SEQ ID NOs: 1 or 3.

The invention also pertains to isolated nucleic acids comprising anucleotide sequence encoding ACE-2 polypeptides, variants and/orequivalents of such nucleic acids. The term equivalent is understood toinclude nucleotide sequences encoding functionally equivalent ACE-2polypeptides or functionally equivalent peptides having an activity ofan ACE-2 protein such as described herein. Equivalent nucleotidesequences will include sequences that differ by one or more nucleotidesubstitution, addition or deletion, such as allelic variants; and will,therefore, include sequences that differ from the nucleotide sequence ofthe ACE-2 gene shown in SEQ ID NOs: 1 or 3 due to the degeneracy of thegenetic code.

Preferred variant ACE-2 nucleic acids are those described in theExamples, such as nucleic acids comprising one or more of SEQ ID NO:87,89, 91, 93, and 95.

Preferred nucleic acids are vertebrate ACE-2 nucleic acids. Particularlypreferred vertebrate ACE-2 nucleic acids are mammalian. Regardless ofspecies, particularly preferred ACE-2 nucleic acids encode polypeptidesthat are at least 70%, 80%, 90%, or 95% similar or identical to an aminoacid sequence of a vertebrate ACE-2 protein. In one embodiment, thenucleic acid is a cDNA encoding a polypeptide having at least onebio-activity of the subject ACE-2 polypeptide. Preferably, the nucleicacid includes all or a portion of the nucleotide sequence correspondingto the nucleic acid of SEQ ID No 1 or 3.

Still other preferred nucleic acids of the present invention encode anACE-2 polypeptide which is comprised of at least 2, 5, 10, 25, 50, 100,150 or 200 amino acid residues. For example, such nucleic acids cancomprise about 50, 60, 70, 80, 90, or 100 base pairs. Also within thescope of the invention are nucleic acid molecules for use asprobes/primer or antisense molecules (i.e. noncoding nucleic acidmolecules), which can comprise at least about 6, 12, 20, 30, 50, 60, 70,80, 90 or 100 base pairs in length.

Another aspect of the invention provides a nucleic acid which hybridizesunder stringent conditions to a nucleic acid represented by SEQ ID NOs:1 or 3 or complement thereof or the nucleic acid having ATCC DesignationNo. 209510. In a particularly preferred embodiment, an ACE-2 nucleicacid of the present invention will bind to one of SEQ ID NOs: 1 or 3 orcomplement thereof under high stringency conditions. As used herein, theterm “hybridizes under stringent conditions” is intended to describeconditions for hybridization and washing under which nucleotidesequences that are significantly identical or homologous to each otherremain hybridized to each other. Preferably, the conditions are suchthat sequences at least about 70%, more preferably at least about 80%,even more preferably at least about 85% or 90% identical to each otherremain hybridized to each other. Such stringent conditions are known tothose skilled in the art and can be found in Current Protocols inMolecular Biology, Ausubel et al., eds., John Wiley & Sons, Inc. (1995),sections 2, 4 and 6. Additional stringent conditions can be found inMolecular Cloning: A Laboratory Manual, Sambrook et al., Cold SpringHarbor Press, Cold Spring Harbor, N.Y. (1989), chapters 7, 9 and 11. Apreferred, non-limiting example of stringent hybridization conditionsincludes hybridization in 4× sodium chloride/sodium citrate (SSC), atabout 65-70° C. (or hybridization in 4×SSC plus 50% formamide at about42-50° C.) followed by one or more washes in 1×SSC, at about 65-70° C. Apreferred, non-limiting example of highly stringent hybridizationconditions includes hybridization in 1×SSC, at about 65-70° C. (orhybridization in 1×SSC plus 50% formamide at about 42-50° C.) followedby one or more washes in 0.3×SSC, at about 65-70° C. A preferred,non-limiting example of reduced stringency hybridization conditionsincludes hybridization in 4×SSC, at about 50-60° C. (or alternativelyhybridization in 6×SSC plus 50% formamide at about 40-45° C.) followedby one or more washes in 2×SSC, at about 50-60° C. Ranges intermediateto the above-recited values, e.g., at 65-70° C. or at 42-50° C. are alsointended to be encompassed by the present invention. SSPE (1×SSPE is0.15M NaCl, 10 mM NaH₂PO₄, and 1.25 mM EDTA, pH 7.4) can be substitutedfor SSC (1×SSC is 0.15M NaCl and 15 mM sodium citrate) in thehybridization and wash buffers; washes are performed for 15 minutes eachafter hybridization is complete. The hybridization temperature forhybrids anticipated to be less than 50 base pairs in length should be5-10° C. less than the melting temperature (T_(m)) of the hybrid, whereT_(m) is determined according to the following equations. For hybridsless than 18 base pairs in length, T_(m)(° C.)=2(# of A+T bases)+4(# ofG+C bases). For hybrids between 18 and 49 base pairs in length, T_(m)(°C.)=81.5+16.6(log₁₀[Na⁺])+0.41 (% G+C)−(600/N), where N is the number ofbases in the hybrid, and [Na⁺] is the concentration of sodium ions inthe hybridization buffer ([Na⁺] for 1×SSC=0.165 M). It will also berecognized by the skilled practitioner that additional reagents may beadded to hybridization and/or wash buffers to decrease non-specifichybridization of nucleic acid molecules to membranes, for example,nitrocellulose or nylon membranes, including but not limited to blockingagents (e.g., BSA or salmon or herring sperm carrier DNA), detergents(e.g., SDS), chelating agents (e.g., EDTA), Ficoll, PVP and the like.When using nylon membranes, in particular, an additional preferred,non-limiting example of stringent hybridization conditions ishybridization in 0.25-0.5M NaH₂PO₄, 7% SDS at about 65° C., followed byone or more washes at 0.02M NaH₂PO₄, 1% SDS at 65° C., see e.g., Churchand Gilbert (1984) Proc. Natl. Acad. Sci. USA 81:1991-1995, (oralternatively 0.2×SSC, 1% SDS).

Nucleic acids having a sequence that differs from the nucleotidesequences shown in one of SEQ ID NOs: 1 or 3 or complement thereof dueto degeneracy in the genetic code are also within the scope of theinvention. Such nucleic acids encode functionally equivalent peptides(i.e., a peptide having a biological activity of an ACE-2 polypeptide)but differ in sequence from the sequence shown in the sequence listingdue to degeneracy in the genetic code. For example, a number of aminoacids are designated by more than one triplet. Codons that specify thesame amino acid, or synonyms (for example, CAU and CAC each encodehistidine) may result in “silent” mutations which do not affect theamino acid sequence of an ACE-2 polypeptide. However, it is expectedthat DNA sequence polymorphisms that do lead to changes in the aminoacid sequences of the subject ACE-2 polypeptides will exist amongmammals. One skilled in the art will appreciate that these variations inone or more nucleotides (e.g., up to about 3-5% of the nucleotides) ofthe nucleic acids encoding polypeptides having an activity of an ACE-2polypeptide may exist among individuals of a given species due tonatural allelic variation.

Nucleic acids of the invention can encode one or more of the followingdomains of an ACE-2 protein: the signal peptide, the extracellulardomain comprising the zinc binding domain, the transmembrane domain, andthe cytoplasmic domain. The amino acid sequences of these domains inhuman ACE-2 (SEQ ID NO:2) and the position of the nucleotide sequence inSEQ ID NO:1 encoding these domains are indicated in Table I: TABLE IPosition of Domains in Human ACE-2 Domain Nucleotides Amino acids signalsequence  82-135  1-18 extracellular domain  136-2301  19-740 min. zincbinding domain 1201-1215 374-378 transmembrane domain 2302-2376 741-765cytoplasmic domain 2377-2496 766-805

The polynucleotide sequence of the present invention may encode a matureform of the ACE-2, i.e., a form of ACE-2 which does not comprise theleader peptide, e.g., an ACE-2 protein which does not comprise aboutamino acids 1-18 of SEQ ID NO:2. For example, a preferred nucleic acidof the invention comprises at least a portion of a nucleotide sequenceencoding ACE-2, but does not include about nucleotides 82-135 of SEQ IDNO:1. The mature form of an ACE-2 polypeptide can be a secreted ACE-2polypeptide or a membrane bound ACE-2 polypeptide. In fact, ACE has beenfound in the form of a membrane enzyme at the surface of the vascularendothelial cells and renal epithelial cells. Alternatively, ACE hasalso been observed to be a secreted protein, and has been found, e.g.,in plasma (see, e.g., Erdos et al. (1987) Lab. Invest. 56:345, Cardwellet al. (1976) Science 191:1050; and Ryan et al. (1976) Tissue Cell8:125). Thus, nucleic acids encoding secreted as well as membrane boundforms of ACE-2 proteins are within the scope of the invention.

In the case of ACE proteins, it has been reported that a soluble form ofthe enzyme results from proteolytic cleavage by a specific enzyme termed“secretase” (Parvathy et al. (1997) Biochem. J. 327:37). Thus, it ispossible that a similar mechanism of solubilization of ACE-2 proteinsoccur. Accordingly, in cases in which the ACE-2 protein is desired as apurely membrane form as opposed to a soluble form, it may be preferableto change the nucleotide sequence of ACE-2 such that it does not encodea site recognizable and cleavable by a secretase.

A recombinant soluble form of ACE-2 can be produced, e.g, by deleting atleast a portion of the transmembrane domain which spans amino acids741-765 of SEQ ID NO:2, such that the protein is not capable to localizeitself to a cell membrane. Thus, nucleic acids of the invention includethose which encode at least a portion of an ACE-2 protein, but whichlacks a portion from about nucleotide 2302 to about nucleotide 2376 ofSEQ ID NO:1. For example, a preferred nucleic acid encoding a solublehuman ACE-2 protein comprises a nucleotide sequence from aboutnucleotide 136 to about nucleotide 2301 of SEQ ID NO:1. Preferredsoluble ACE-2 proteins comprise at least a portion of the extracellulardomain of ACE-2 which corresponds to about amino acid 19 to about aminoacid 740 of SEQ ID NO:2 and is encoded by the nucleotide sequence fromabout nucleotide 136 to about nucleotide 2301 of SEQ ID NO:1.

The polynucleotide sequence may also encode a leader sequence, e.g., thenatural leader sequence or a heterologous leader sequence. Human ACE-2has a leader sequence from amino acid 1 to amino acid 18 of SEQ ID NO:2.Accordingly, the polynucleotide may encode the natural ACE-2 leadersequence. Alternatively, the nucleic acid can be engineered such thatthe natural leader sequence is deleted and a heterologous leadersequence inserted in its place. The term “leader sequence” is usedinterchangeably herein with the term “signal peptide”. For example, thedesired DNA sequence may be fused in the same reading frame to a DNAsequence which aids in expression and secretion of the polypeptide fromthe host cell, for example, a leader sequence which functions as asecretory sequence for controlling transport of the polypeptide from thecell. The protein having a leader sequence is a preprotein and may havethe leader sequence cleaved by the host cell to form the mature form ofthe protein.

The polynucleotide of the present invention may also be fused in frameto a marker sequence, also referred to herein as “Tag sequence” encodinga “Tag peptide”, which allows for marking and/or purification of thepolypeptide of the present invention. In a preferred embodiment, themarker sequence is a hexahistidine tag, e.g., supplied by a PQE-9vector. Numerous other Tag peptides are available commercially. Otherfrequently used Tags include myc-epitopes (e.g., see Ellison et al.(1991) J Biol Chem 266:21150-21157) which includes a 10-residue sequencefrom c-myc, the pFLAG system (International Biotechnologies, Inc.), thepEZZ-protein A system (Pharmacia, N.J.), and a 16 amino acid portion ofthe Haemophilus influenza hemagglutinin protein. Furthermore, anypolypeptide can be used as a Tag so long as a reagent, e.g., an antibodyinteracting specifically with the Tag polypeptide is available or can beprepared or identified.

In another embodiment, a fusion gene coding for a purification leadersequence, such as a poly-(His)/enterokinase cleavage site sequence atthe N-terminus of the desired portion of the recombinant protein, canallow purification of the expressed fusion protein by affinitychromatography using a Ni²⁺ metal resin. The purification leadersequence can then be subsequently removed by treatment with enterokinaseto provide the purified protein (e.g., see Hochuli et al. (1987) J.Chromatography 411:177; and Janknecht et al. PNAS 88:8972).

Techniques for making fusion genes are known to those skilled in theart. Essentially, the joining of various DNA fragments coding fordifferent polypeptide sequences is performed in accordance withconventional techniques, employing blunt-ended or stagger-ended terminifor ligation, restriction enzyme digestion to provide for appropriatetermini, filling-in of cohesive ends as appropriate, alkalinephosphatase treatment to avoid undesirable joining, and enzymaticligation. In another embodiment, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, PCR amplification of gene fragments can be carried outusing anchor primers which give rise to complementary overhangs betweentwo consecutive gene fragments which can subsequently be annealed togenerate a chimeric gene sequence (see, for example, Current Protocolsin Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).

Other preferred ACE-2 fusion proteins include ACE-2-immunoglobulin(ACE-2-Ig) polypeptides. The ACE-2-Ig polypeptide can comprise theentire extracellular domain of ACE-2, e.g, human ACE-2, or a variantthereof. For example, an ACE-2-Ig polypeptide can comprise an amino acidsequences from about amino acid 1 to about amino acid 740 of SEQ IDNO:2. A nucleic acid encoding an ACE-2Ig fusion protein can comprise,e.g., about nucleotides 82 to 2301 of SEQ ID NO:1 fused in frame to anucleic acid encoding a constant Ig chain. ACE-2-Ig fusion proteins canbe prepared as described e.g., in U.S. Pat. No. 5,434,131.

As indicated by the examples set out below, ACE-2 protein-encodingnucleic acids can be obtained from mRNA present in any of a number ofeukaryotic cells, e.g., from cardiac tissue or kidney. It should also bepossible to obtain nucleic acids encoding ACE-2 polypeptides of thepresent invention from genomic DNA from both adults and embryos. Forexample, a gene encoding an ACE-2 protein can be cloned from either acDNA or a genomic library in accordance with protocols described herein,as well as those generally known to persons skilled in the art. cDNAencoding an ACE-2 protein can be obtained by isolating total mRNA from acell, e.g., a vertebrate cell, a mammalian cell, or a human cell,including embryonic cells. Double stranded cDNAs can then be preparedfrom the total mRNA, and subsequently inserted into a suitable plasmidor bacteriophage vector using any one of a number of known techniques.The gene encoding an ACE-2 protein can also be cloned using establishedpolymerase chain reaction techniques in accordance with the nucleotidesequence information provided by the invention. The nucleic acid of theinvention can be DNA or RNA or analogs thereof. A preferred nucleic acidis a cDNA represented by a sequence selected from the group consistingof SEQ ID NOs: 1 or 3.

Preferred nucleic acids encode a vertebrate ACE-2 polypeptide comprisingan amino acid sequence that is at least about 60% homologous, morepreferably at least about 70% homologous and most preferably at leastabout 80% homologous with amino acid sequence contained in SEQ ID No: 2.Nucleic acids which encode polypeptides at least about 90%, morepreferably at least about 95%, and most preferably at least about 98-99%homology with an amino acid sequence represented in SEQ ID No: 2 arealso within the scope of the invention. In one embodiment, the nucleicacid is a cDNA encoding a peptide having at least one activity of thesubject vertebrate ACE-2 polypeptide. Preferably, the nucleic acidincludes all or a portion of the nucleotide sequence corresponding tothe coding region of SEQ ID NOs:1 and 3.

Preferred nucleic acids encode a bioactive fragment of a vertebrateACE-2 polypeptide comprising an amino acid sequence at least about 60%homologous or identical, more preferably at least about 70% homologousor identical and most preferably at least about 80% homologous oridentical with an amino acid sequence of SEQ ID No: 2. Nucleic acidswhich encode polypeptides which are at least about 90%, more preferablyat least about 95%, and most preferably at least about 98-99% homologousor identical, with an amino acid sequence represented in SEQ ID No: 2are also within the scope of the invention.

Bioactive fragments of ACE-2 polypeptides can be polypeptides having oneor more of the following biological activities: the capability tocatalyze an enzymatic reaction, e.g., the hydrolysis of a targetpeptide, such as the hydrolysis of an angiotensin I peptide intoAng.(1-9) peptide, the hydrolysis of a kinin or derivative thereof,e.g., bradykinin or hydrolysis of kinetensin; binding to a metal ion,e.g., zinc, interacting with a substrate, e.g., angiotensin I, a kinin,or kinetensin. A bioactive fragment of an ACE-2 polypeptide can also bea polypeptide having an analgesic activity, an anti-inflammatoryactivity and/or an anti-allergenic activity, the capability to modulatecell growth, to interact with another molecule, e.g., a target peptideor a receptor. Assays for determining whether an ACE-2 polypeptide hasany of these or other biological activities are known in the art and arefurther described herein.

Nucleic acids encoding proteins having an ACE-2 activity include nucleicacids comprising a nucleotide sequence encoding a zinc binding domain,such as the zinc binding domain of ACE-2 consisting of about amino acids374-378 of SEQ ID NO:2. Such a nucleic acid can be represented by thegeneric formula: X-(ZBD)-Y, wherein ZBD represents nucleotides 1201-1215of SEQ ID NO:1, and X and Y represent a certain number of nucleotideslocated 5′ and 3′ of the ZBD, respectively. For example, a nucleic acidof the invention can comprise nucleotides 1201-1215 of SEQ ID NO:1 and Xand Y selected from any of 0, 10, 20, 30, 50, 100, 150, 200, 300, 400,500, 600, 700, 800, 900, or about 1000 nucleotides. Alternatively, ZBDcan represent the nucleotide sequence from nucleotide 1192 to 1221 or1195 to 1224 of SEQ ID NO:1 or any sequence substantially similarthereto, or comprising one or more nucleotides at the 5′ and/or 3′ end.Another preferred ZBD is an extended ZBD, i.e., comprising nucleotidesencoding amino acids which are located outside of amino acids 371-380,but which are functionally active. For example, a preferred nucleic acidcomprises a nucleotide sequence from about nucleotide 1201 to aboutnucleotide 1331 of SEQ ID NO:1 and encoding all the amino acids whicheither contact the zinc atom or which are involved in catalysis. Thesenucleic acids preferably encode a protein having a biological activity,e.g., the capability to catalyze the hydrolysis of a peptide.Alternatively such polypeptides are devoid of biological activity.Accordingly, the invention provides nucleic acids comprising nucleotidesequence from about nucleotide 1201 to about nucleotide 1331 of SEQ IDNO:1, in which codons encoding residues 374, 375, 378, 402, 406, and/or417 are mutated.

Nucleic acids encoding modified forms or mutant forms of ACE-2 alsoinclude those encoding ACE-2 proteins having mutated glycosylationsites, such that either the encoded ACE-2 protein is not glycosylated,partially glycosylated and/or has a modified glycosylation pattern.Seven potential N-linked glycosylation sites have been identified inhuman ACE-2 and these are located at amino acids 53, 90, 103, 322, 432,546, and 690 in SEQ ID NO:2. Glycosylation sites, N-glycosylation orO-glycosylation sites can also be added to the protein. Amino acidsequence motifs required for the attachment of a sugar unit are wellknown in the art.

Other preferred nucleic acids of the invention include nucleic acidsencoding derivatives of ACE-2 polypeptides which lack one or morebiological activities of ACE-2 polypeptides. For example, the inventionprovides derivatives of ACE-2 polypeptides having an anti-inflammatoryactivity but which are essentially incapable of hydrolyzing angiotensinI. Such nucleic acids can be obtained, e.g., by a first round ofscreening of libraries for the presence or absence of a first activityand a second round of screening for the presence or absence of anotheractivity.

Also within the scope of the invention are nucleic acids encoding splicevariants or nucleic acids representing transcripts synthesized from analternative transcriptional initiation site, such as those whosetranscription was initiated from a site in an intron., as is the casewith the testicular ACE mRNAs. Such homologs can be cloned byhybridization or PCR, as further described herein.

In preferred embodiments, the ACE-2 nucleic acids can be modified at thebase moiety, sugar moiety or phosphate backbone to improve, e.g., thestability, hybridization, or solubility of the molecule. For example,the deoxyribose phosphate backbone of the nucleic acids can be modifiedto generate peptide nucleic acids (see Hyrup B. et al. (1996) Bioorganic& Medicinal Chemistry 4 (1): 5-23). As used herein, the terms “peptidenucleic acids” or “PNAS” refer to nucleic acid mimics, e.g., DNA mimics,in which the deoxyribose phosphate backbone is replaced by apseudopeptide backbone and only the four natural nucleobases areretained. The neutral backbone of PNAs has been shown to allow forspecific hybridization to DNA and RNA under conditions of low ionicstrength. The synthesis of PNA oligomers can be performed using standardsolid phase peptide synthesis protocols as described in Hyrup B. et al.(1996) supra; Perry-O'Keefe et al. PNAS 93: 14670-675.

PNAs of ACE-2 can be used in therapeutic and diagnostic applications andare further described herein in section 4.3.2. Such modified nucleicacids can be used as antisense or antigene agents for sequence-specificmodulation of gene expression or in the analysis of single base pairmutations in a gene by, e.g., PNA directed PCR clamping or as probes orprimers for DNA sequence and hybridization (Hyrup B. et al (1996) supra;Perry-O'Keefe supra).

PNAs of ACE-2 can further be modified, e.g., to enhance their stabilityor cellular uptake, e.g., by attaching lipophilic or other helper groupsto the ACE-2 PNA, by the formation of PNA-DNA chimeras, or by the use ofliposomes or other techniques of drug delivery known in the art. ACE-2PNAs can also be linked to DNA as described, e.g., in Hyrup B. (1996)supra and Finn P. J. et al. (1996) Nucleic Acids Research 24 (17):3357-63. For example, a DNA chain can be synthesized on a solid supportusing standard phosphoramidite coupling chemistry and modifiednucleoside analogs, e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidinephosphoramidite, can be used as a between the PNA and the 5′ end of DNA(Mag, M. et al. (1989) Nucleic Acid Res. 17: 5973-88). PNA monomers arethen coupled in a stepwise manner to produce a chimeric molecule with a5′ PNA segment and a 3′ DNA segment (Finn P. J. et al. (1996) supra).Alternatively, chimeric moleclues can be synthesized with a 5′ DNAsegment and a 3′ PNA segment (Peterser, K. H. et al. (1975) BioorganicMed Chem. Lett. 5: 1119-11124).

In other embodiments, ACE-2 nucleic acids may include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents that facilitate transport across the cell membrane asdescribed in section 4.3.2. herein.

3.1. Probes and Primers

The nucleotide sequences determined from the cloning of ACE-2 genes frommammalian organisms will further allow for the generation of probes andprimers designed for use in identifying and/or cloning ACE-2 homologs inother cell types, e.g., from other tissues, as well as ACE-2 homologsfrom other mammalian organisms. For instance, the present invention alsoprovides a probe/primer comprising a substantially purifiedoligonucleotide, which oligonucleotide comprises a region of nucleotidesequence that hybridizes under stringent conditions to at leastapproximately 12, preferably 25, more preferably 40, 50 or 75consecutive nucleotides of sense or anti-sense sequence selected fromthe group consisting of SEQ ID No: 1 or 3 or naturally occurring mutantsthereof. For instance, primers based on the nucleic acid represented inSEQ ID NOs:1 or 3 can be used in PCR reactions to clone ACE-2 homologs.

Likewise, probes based on the subject ACE-2 sequences can be used todetect transcripts or genomic sequences encoding the same or homologousproteins, for use, e.g, in prognostic or diagnostic assays (furtherdescribed below). In preferred embodiments, the probe further comprisesa label group attached thereto and able to be detected, e.g., the labelgroup is selected from amongst radioisotopes, fluorescent compounds,enzymes, and enzyme co-factors. Preferred probes for detectingpolymorphisms are described in the Examples.

Probes and primers can be prepared and modified as described in theother sections herein relating to nucleic acids.

3.2 Antisense, Ribozyme and Triplex Techniques

Another aspect of the invention relates to the use of the isolatednucleic acid in “antisense” therapy. As used herein, “antisense” therapyrefers to administration or in situ generation of oligonucleotidemolecules or their derivatives which specifically hybridize (e.g., bind)under cellular conditions, with the cellular mRNA and/or genomic DNAencoding one or more of the subject ACE-2 proteins so as to inhibitexpression of that protein, e.g., by inhibiting transcription and/ortranslation. The binding may be by conventional base paircomplementarity, or, for example, in the case of binding to DNAduplexes, through specific interactions in the major groove of thedouble helix. In general, “antisense” therapy refers to the range oftechniques generally employed in the art, and includes any therapy whichrelies on specific binding to oligonucleotide sequences.

An antisense construct of the present invention can be delivered, forexample, as an expression plasmid which, when transcribed in the cell,produces RNA which is complementary to at least a unique portion of thecellular mRNA which encodes an ACE-2 protein. Alternatively, theantisense construct is an oligonucleotide probe which is generated exvivo and which, when introduced into the cell causes inhibition ofexpression by hybridizing with the mRNA and/or genomic sequences of anACE-2 gene. Such oligonucleotide probes are preferably modifiedoligonucleotides which are resistant to endogenous nucleases, e.g.,exonucleases and/or endonucleases, and are therefore stable in vivo.Exemplary nucleic acid molecules for use as antisense oligonucleotidesare phosphoramidate, phosphothioate and methylphosphonate analogs of DNA(see also U.S. Pat. Nos. 5,176,996; 5,264,564; and 5,256,775).Additionally, general approaches to constructing oligomers useful inantisense therapy have been reviewed, for example, by Van der Krol etal. (1988) BioTechniques 6:958-976; and Stein et al. (1988) Cancer Res48:2659-2668. With respect to antisense DNA, oligodeoxyribonucleotidesderived from the translation initiation site, e.g., between the −10 and+10 regions of the ACE-2 nucleotide sequence of interest, are preferred.

Antisense approaches involve the design of oligonucleotides (either DNAor RNA) that are complementary to ACE-2 mRNA. The antisenseoligonucleotides will bind to the ACE-2 mRNA transcripts and preventtranslation. Absolute complementarity, although preferred, is notrequired. In the case of double-stranded antisense nucleic acids, asingle strand of the duplex DNA may thus be tested, or triplex formationmay be assayed. The ability to hybridize will depend on both the degreeof complementarity and the length of the antisense nucleic acid.Generally, the longer the hybridizing nucleic acid, the more basemismatches with an RNA it may contain and still form a stable duplex (ortriplex, as the case may be). One skilled in the art can ascertain atolerable degree of mismatch by use of standard procedures to determinethe melting point of the hybridized complex.

Oligonucleotides that are complementary to the 5′ end of the mRNA, e.g.,the 5′ untranslated sequence up to and including the AUG initiationcodon, should work most efficiently at inhibiting translation. However,sequences complementary to the 3′ untranslated sequences of mRNAs haverecently been shown to be effective at inhibiting translation of mRNAsas well. (Wagner, R. 1994. Nature 372:333). Therefore, oligonucleotidescomplementary to either the 5′ or 3′ untranslated, non-coding regions ofan ACE-2 gene could be used in an antisense approach to inhibittranslation of endogenous ACE-2 mRNA. Oligonucleotides complementary tothe 5′ untraslated region of the mRNA should include the complement ofthe AUG start codon. Antisense oligonucleotides complementary to mRNAcoding regions are less efficient inhibitors of translation but couldalso be used in accordance with the invention. Whether designed tohybridize to the 5′, 3′ or coding region of ACE-2 mRNA, antisensenucleic acids should be at least six nucleotides in length, and arepreferably less that about 100 and more preferably less than about 50,25, 17 or 10 nucleotides in length.

Regardless of the choice of target sequence, it is preferred that invitro studies are first performed to quantitate the ability of theantisense oligonucleotide to quantitate the ability of the antisenseoligonucleotide to inhibit gene expression. It is preferred that thesestudies utilize controls that distinguish between antisense geneinhibition and nonspecific biological effects of oligonucleotides. It isalso preferred that these studies compare levels of the target RNA orprotein with that of an internal control RNA or protein. Additionally,it is envisioned that results obtained using the antisenseoligonucleotide are compared with those obtained using a controloligonucleotide. It is preferred that the control oligonucleotide is ofapproximately the same length as the test oligonucleotide and that thenucleotide sequence of the oligonucleotide differs from the antisensesequence no more than is necessary to prevent specific hybridization tothe target sequence.

The oligonucleotides can be DNA or RNA or chimeric mixtures orderivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide may includeother appended groups such as peptides (e.g., for targeting host cellreceptors), or agents facilitating transport across the cell membrane(see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. U.S.A.86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648-652;PCT Publication No. W088/09810, published Dec. 15, 1988) or theblood-brain barrier (see, e.g., PCT Publication No. W089/10134,published Apr. 25, 1988), hybridization-triggered cleavage agents. (See,e.g., Krol et al., 1988, BioTechniques 6:958-976) or intercalatingagents. (See, e.g., Zon, 1988, Pharm. Res. 5:539-549). To this end, theoligonucleotide may be conjugated to another molecule, e.g., a peptide,hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

The antisense oligonucleotide may comprise at least one modified basemoiety which is selected from the group including but not limited to5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine,5-(carboxyhydroxytiethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w,and 2,6-diaminopurine.

The antisense oligonucleotide may also comprise at least one modifiedsugar moiety selected from the group including but not limited toarabinose, 2-fluoroarabinose, xylulose, and hexose.

The antisense oligonucleotide can also contain a neutral peptide-likebackbone. Such molecules are termed peptide nucleic acid (PNA)-oligomersand are described, e.g., in Perry-O'Keefe et al. (1 996) Proc. Natl.Acad. Sci. U.S.A. 93:14670 and in Eglom et al. (1993) Nature 365:566.One advantage of PNA oligomers is their capability to bind tocomplementary DNA essentially independently from the ionic strength ofthe medium due to the neutral backbone of the DNA. In yet anotherembodiment, the antisense oligonucleotide comprises at least onemodified phosphate backbone selected from the group consisting of aphosphorothioate, a phosphorodithioate, a phosphoramidothioate, aphosphoramidate, a phosphordiamidate, a methylphosphonate, an alkylphosphotriester, and a formacetal or analog thereof.

In yet a further embodiment, the antisense oligonucleotide is anα-anomeric oligonucleotide. An a-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a2′-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330).

Oligonucleotides of the invention may be synthesized by standard methodsknown in the art, e.g., by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides may be synthesized by themethod of Stein et al. (1988, Nucl. Acids Res. 16:3209),methylphosphonate olgonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.U.S.A. 85:7448-7451), etc.

While antisense nucleotides complementary to the ACE-2 coding regionsequence can be used, those complementary to the transcribeduntranslated region and to the region comprising the initiatingmethionine are most preferred.

The antisense molecules can be delivered to cells which express ACE-2 invivo. A number of methods have been developed for delivering antisenseDNA or RNA to cells; e.g., antisense molecules can be injected directlyinto the tissue site, or modified antisense molecules, designed totarget the desired cells (e.g., antisense linked to peptides orantibodies that specifically bind receptors or antigens expressed on thetarget cell surface) can be administered systematically.

However, it may be difficult to achieve intracellular concentrations ofthe antisense sufficient to suppress translation on endogenous mRNAs incertain instances. Therefore a preferred approach utilizes a recombinantDNA construct in which the antisense oligonucleotide is placed under thecontrol of a strong pol III or pol II promoter. The use of such aconstruct to transfect target cells in the patient will result in thetranscription of sufficient amounts of single stranded RNAs that willform complementary base pairs with the endogenous ACE-2 transcripts andthereby prevent translation of the ACE-2 mRNA. For example, a vector canbe introduced in vivo such that it is taken up by a cell and directs thetranscription of an antisense RNA. Such a vector can remain episomal orbecome chromosomally integrated, as long as it can be transcribed toproduce the desired antisense RNA. Such vectors can be constructed byrecombinant DNA technology methods standard in the art. Vectors can beplasmid, viral, or others known in the art, used for replication andexpression in mammalian cells. Expression of the sequence encoding theantisense RNA can be by any promoter known in the art to act inmammalian, preferably human cells. Such promoters can be inducible orconstitutive. Such promoters include but are not limited to: the SV40early promoter region (Bernoist and Chambon, 1981, Nature 290:304-310),the promoter contained in the 3′ long terminal repeat of Rous sarcomavirus (Yamamoto et al., 1980, Cell 22:787-797), the herpes thymidinekinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A.78:1441-1445), the regulatory sequences of the metallothionein gene(Brinster et al, 1982, Nature 296:39-42), etc. Any type of plasmid,cosmid, YAC or viral vector can be used to prepare the recombinant DNAconstruct which can be introduced directly into the tissue site.Alternatively, viral vectors can be used which selectively infect thedesired tissue, in which case administration may be accomplished byanother route (e.g., systematically).

Ribozyme molecules designed to catalytically cleave ACE-2 mRNAtrnscripts can also be used to prevent translation of ACE-2 mRNA andexpression of ACE-2 (See, e.g., PCT International PublicationWO90/11364, published Oct. 4, 1990; Sarver et al., 1990, Science247:1222-1225 and U.S. Pat. No. 5,093,246). While ribozymes that cleavemRNA at site specific recognition sequences can be used to destroy ACE-2mRNAs, the use of hammerhead ribozymes is preferred. Hammerheadribozymes cleave mRNAs at locations dictated by flanking regions thatform complementary base pairs with the target mRNA. The sole requirementis that the target mRNA have the following sequence of two bases:5′-UG-3′. The construction and production of hammerhead ribozymes iswell known in the art and is described more fully in Haseloff andGerlach, 1988, Nature, 334:585-591. There are a number of potentialhammerhead ribozyme cleavage sites within the nucleotide sequence ofhuman ACE-2 cDNA (FIG. 1). Preferably the ribozyrne is engineered sothat the cleavage recognition site is located near the 5′ end of theACE-2 mRNA; i.e., to increase efficiency and minimize the intracellularaccumulation of non-functional mRNA transcripts.

The ribozymes of the present invention also include RNAendoribonucleases (hereinafter “Cech-type ribozymes”) such as the onewhich occurs naturally in Tetrahymena thermophila (known as the IVS, orL-19 IVS RNA) and which has been extensively described by Thomas Cechand collaborators (Zaug, et al., 1984, Science, 224:574-578; Zaug andCech, 1986, Science, 231:470-475; Zaug, et al., 1986, Nature,324:429-433; published International patent application No. WO88/04300by University Patents Inc.; Been and Cech, 1986, Cell, 47:207-216). TheCech-type ribozymes have an eight base pair active site which hybridizesto a target RNA sequence whereafter cleavage of the target RNA takesplace. The invention encompasses those Cech-type ribozymes which targeteight base-pair active site sequences that are present in an ACE-2 gene.

As in the antisense approach, the ribozymes can be composed of modifiedoligonucleotides (e.g., for improved stability, targeting, etc.) andshould be delivered to cells which express the ACE-2 gene in vivo. Apreferred method of delivery involves using a DNA construct “encoding”the robozyme under the control of a strong constitutive pol III or polII promoter, so that transfected cells will produce sufficientquantities of the ribozyme to destroy endogenous ACE-2 messages andinhibit translation. Because ribozymes unlike antisense molecules, arecatalytic, a lower intracellular concentration is required forefficiency.

Endogenous ACE-2 gene expression can also be reduced by inactivating or“knocking out” the ACE-2 gene or its promoter using targeted homologousrecombination. (E.g., see Smithies et al., 1985, Nature 317:230-234;Thomas & Capecchi, 1987, Cell 51:503-512; Thompson et al., 1989 Cell5:313-321; each of which is incorporated by reference herein in itsentirety). For example, a mutant, non-functional ACE-2 (or a completelyunrelated DNA sequence) flanked by DNA homologous to the endogenousACE-2 gene (either the coding regions or regulatory regions of the ACE-2gene) can be used, with or without a selectable marker and/or a negativeselectable marker, to transfect cells that express ACE-2 in vivo.Insertion of the DNA construct, via targeted homologous recombination,results in inactivation of the ACE-2 gene. Such approaches areparticularly suited in the agricultural field where modifications to ES(embryonic stem) cells can be used to generate animal offspring with aninactive ACE-2 (e.g., see Thomas & Capecchi 1987 and Thompson 1989,supra). However this approach can be adapted for use in humans providedthe recombinant DNA constructs are directly administered or targeted tothe required site in vivo using appropriate viral vectors.

Alternatively, endogenous ACE-2 gene expression can be reduced bytargeting deoxyribonucleotide sequences complementary to the regulatoryregion of the ACE-2 gene (i.e., the ACE-2 promoter and/or enhancers) toform triple helical structures that prevent transcription of the ACE-2gene in target cells in the body. (See generally, Helene, C. 1991,Anticancer Drug Des., 6(6):569-84; Helene, C., et al., 1992, Ann. N.Y.Acad. Sci., 660:27-36; and Maher, L. J., 1992, Bioassays 14(12):807-15).

Nucleic acid molecules to be used in triple helix formation for theinhibition of transcription are preferably single stranded and composedof deoxyribonucleotides. The base composition of these oligonucleotidesshould promote triple helix formation via Hoogsteen base pairing rules,which generally require sizable stretches of either purines orpyrimidines to be present on one strand of a duplex. Nucleotidesequences may be pyrimidine-based, which will result in TAT and CGCtriplets across the three associated strands of the resulting triplehelix. The pyrimidine-rich molecules provide base complementarity to apurine-rich region of a single strand of the duplex in a parallelorientation to that strand. In addition, nucleic acid molecules may bechosen that are purine-rich, for example, containing a stretch of Gresidues. These molecules will form a triple helix with a DNA duplexthat is rich in GC pairs, in which the majority of the purine residuesare located on a single strand of the targeted duplex, resulting in CGCtriplets across the three strands in the triplex.

Alternatively, the potential sequences that can be targeted for triplehelix formation may be increased by creating a so called “switchback”nucleic acid molecule. Switchback molecules are synthesized in analternating 5′-3′, 3′-5′ manner, such that they base pair with first onestrand of a duplex and then the other, eliminating the necessity for asizable stretch of either purines or pyrimidines to be present on onestrand of a duplex.

Antisense RNA and DNA, ribozyme, and triple helix molecules of theinvention may be prepared by any method known in the art for thesynthesis of DNA and RNA molecules. These include techniques forchemically synthesizing oligodeoxyribonucleotides andoligoribonucleotides well known in the art such as for example solidphase phosphoramidite chemical synthesis. Alternatively, RNA moleculesmay be generated by in vitro and in vivo transcription of DNA sequencesencoding the antisense RNA molecule. Such DNA sequences may beincorporated into a wide variety of vectors which incorporate suitableRNA polymerase promoters such as the T7 or SP6 polymerase promoters.Alternatively, antisense cDNA constructs that synthesize antisense RNAconstitutively or inducibly, depending on the promoter used, can beintroduced stably into cell lines.

Moreover, various well-known modifications to nucleic acid molecules maybe introduced as a means of increasing intracellular stability andhalf-life. Possible modifications include but are not limited to theaddition of flanking sequences of ribonucleotides ordeoxyribonucleotides to the 5′ and/or 3′ ends of the molecule or the useof phosphorothioate or 2′ O-methyl rather than phosphodiesteraselinkages within the oligodeoxyribonucleotide backbone.

3.3. Vectors Encoding ACE-2 Proteins and ACE-2 Expressing Cells

The invention further provides plasmids and vectors encoding an ACE-2protein, which can be used to express an ACE-2 protein in a host cell.The host cell may be any prokaryotic or eukaryotic cell. Thus, anucleotide sequence derived from the cloning of mammalian ACE-2proteins, encoding all or a selected portion of the full-length protein,can be used to produce a recombinant form of an ACE-2 polypeptide viamicrobial or eukaryotic cellular processes. Ligating the polynucleotidesequence into a gene construct, such as an expression vector, andtransforming or transfecting into hosts, either eukaryotic (yeast,avian, insect or mammalian) or prokaryotic (bacterial) cells, arestandard procedures well known in the art.

Vectors that allow expression of a nucleic acid in a cell are referredto as expression vectors. Typically, expression vectors used forexpressing an ACE-2 protein contain a nucleic acid encoding an ACE-2polypeptide, operably linked to at least one transcriptional regulatorysequence. Regulatory sequences are art-recognized and are selected todirect expression of the subject ACE-2 proteins. Transcriptionalregulatory sequences are described in Goeddel; Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990). In one embodiment, the expression vector includes a recombinantgene encoding a peptide having an agonistic activity of a subject ACE-2polypeptide, or alternatively, encoding a peptide which is anantagonistic form of an ACE-2 protein.

Suitable vectors for the expression of an ACE-2 polypeptide includeplasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids,pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmidsfor expression in prokaryotic cells, such as E. coli.

A number of vectors exist for the expression of recombinant proteins inyeast. For instance, YEP24, YIP5, YEP51, YEP52, pYES2, and YRP17 arecloning and expression vehicles useful in the introduction of geneticconstructs into S. cerevisiae (see, for example, Broach et al. (1983) inExperimental Manipulation of Gene Expression, ed. M. Inouye AcademicPress, p. 83, incorporated by reference herein). These vectors canreplicate in E. coli due the presence of the pBR322 ori, and in S.cerevisiae due to the replication determinant of the yeast 2 micronplasmid. In addition, drug resistance markers such as ampicillin can beused. In an illustrative embodiment, an ACE-2 polypeptide is producedrecombinantly utilizing an expression vector generated by sub-cloningthe coding sequence of one of the ACE-2 genes represented in SEQ IDNOs:1 or 3.

The preferred mammalian expression vectors contain both prokaryoticsequences, to facilitate the propagation of the vector in bacteria, andone or more eukaryotic transcription units that are expressed ineukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo,pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectorsare examples of mammalian expression vectors suitable for transfectionof eukaryotic cells. Some of these vectors are modified with sequencesfrom bacterial plasmids, such as pBR322, to facilitate replication anddrug resistance selection in both prokaryotic and eukaryotic cells.Alternatively, derivatives of viruses such as the bovine papillomavirus(BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can beused for transient expression of proteins in eukaryotic cells. Thevarious methods employed in the preparation of the plasmids andtransformation of host organisms are well known in the art. For othersuitable expression systems for both prokaryotic and eukaryotic cells,as well as general recombinant procedures, see Molecular Cloning ALaboratory Manual, 2^(nd) Ed., ed. by Sambrook, Fritsch and Maniatis(Cold Spring Harbor Laboratory Press: 1989) Chapters 16 and 17.

In some instances, it may be desirable to express the recombinant ACE-2polypeptide by the use of a baculovirus expression system. Examples ofsuch baculovirus expression systems include pVL-derived vectors (such aspVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUW1),and pBlueBac-derived vectors (such as the β-gal containing pBlueBacIII). Production of ACE-2 in this system is further described in theExamples.

When it is desirable to express only a portion of an ACE-2 protein, suchas a form lacking a portion of the N-terminus, i.e. a truncation mutantwhich lacks the signal peptide, it may be necessary to add a start codon(ATG) to the oligonucleotide fragment containing the desired sequence tobe expressed. It is well known in the art that a methionine at theN-terminal position can be enzymatically cleaved by the use of theenzyme methionine aminopeptidase (MAP). MAP has been cloned from E. coli(Ben-Bassat et al. (1987) J. Bacteriol. 169:751-757) and Salmonellatyphimurium and its in vitro activity has been demonstrated onrecombinant proteins (Miller et al. (1987) PNAS 84:2718-1722).Therefore, removal of an N-terminal methionine, if desired, can beachieved either in vivo by expressing ACE-2 derived polypeptides in ahost which produces MAP (e.g., E. coli or CM89 or S. cerevisiae), or invitro by use of purified MAP (e.g., procedure of Miller et al., supra).

Moreover, the gene constructs of the present invention can also be usedas part of a gene therapy protocol to deliver nucleic acids encodingeither an agonistic or antagonistic form of one of the subject ACE-2proteins. Thus, another aspect of the invention features expressionvectors for in vivo or in vitro transfection and expression of an ACE-2polypeptide in particular cell types so as to reconstitute the functionof, or alternatively, abrogate the function of ACE-2 in a tissue. Thiscould be desirable, for example, when the naturally-occurring form ofthe protein is misexpressed or the natural protein is mutated and lessactive.

In addition to viral transfer methods, non-viral methods can also beemployed to cause expression of a subject ACE-2 polypeptide in thetissue of an animal. Most nonviral methods of gene transfer rely onnormal mechanisms used by mammalian cells for the uptake andintracellular transport of macromolecules. In preferred embodiments,non-viral targeting means of the present invention rely on endocyticpathways for the uptake of the subject ACE-2 polypeptide gene by thetargeted cell. Exemplary targeting means of this type include liposomalderived systems, poly-lysine conjugates, and artificial viral envelopes.

In other embodiments transgenic animals, described in more detail belowcould be used to produce recombinant proteins.

4. Polypeptides of the Present Invention

The present invention makes available isolated ACE-2 polypeptides whichare isolated from, or otherwise substantially free of other cellularproteins. The term “substantially free of other cellular proteins” (alsoreferred to herein as “contaminating proteins”) or “substantially pureor purified preparations” are defined as encompassing preparations ofACE-2 polypeptides having less than about 20% (by dry weight)contaminating protein, and preferably having less than about 5%contaminating protein. Functional forms of the subject polypeptides canbe prepared, for the first time, as purified preparations by using acloned gene as described herein.

Preferred ACE-2 proteins of the invention have an amino acid sequencewhich is at least about 60%, 70%, 80%, 85%, 90%, or 95% identical orhomologous to an amino acid sequence of SEQ ID NO:2. Even more preferredACE-2 proteins comprise an amino acid sequence which is at least about97, 98, or 99% homologous or identical to an amino acid sequence of SEQID NO:2. Such proteins can be recombinant proteins, and can be, e.g.,produced in vitro from nucleic acids comprising a nucleotide sequenceset forth in SEQ ID NO:1 or 3, or homologs thereof. For example,recombinant polypeptides preferred by the present invention can beencoded by a nucleic acid, which is at least 85% homologous and morepreferably 90% homologous and most preferably 95% homologous with anucleotide sequence set forth in SEQ ID NOs: 1 or 3. Polypeptides whichare encoded by a nucleic acid that is at least about 98-99% homologouswith the sequence of SEQ ID NOs: 1 or 3 are also within the scope of theinvention.

In a preferred embodiment, an ACE-2 protein of the present invention isa mammalian ACE-2 protein. In a particularly preferred embodiment anACE-2 protein is set forth as SEQ ID No: 2. In another preferredembodiment, the human ACE-2 protein consists of the amino acid sequenceset forth in SEQ ID NO:106, which is identical to SEQ ID NO:2, exceptfor the presence of an aspartic acid at residue 720. In particularlypreferred embodiment, an ACE-2 protein has an ACE-2 bioactivity. It willbe understood that certain post-translational modifications, e.g.,phosphorylation and the like, can increase the apparent molecular weightof the ACE-2 protein relative to the unmodified polypeptide chain.

ACE-2 polypeptides preferably are capable of functioning in one ofeither role of an agonist or antagonist of at least one biologicalactivity of a wild-type (“authentic”) ACE-2 protein of the appendedsequence listing. The term “evolutionarily related to”, with respect toamino acid sequences of ACE-2 proteins, refers to both polypeptideshaving amino acid sequences which have arisen naturally, and also tomutational variants of human ACE-2 polypeptides which are derived, forexample, by combinatorial mutagenesis.

Full length proteins or fragments corresponding to one or moreparticular motifs and/or domains or to arbitrary sizes, for example, atleast 5, 10, 25, 50, 75 and 100, amino acids in length are within thescope of the present invention.

For example, isolated ACE-2 polypeptides can be encoded by all or aportion of a nucleic acid sequence shown in any of SEQ ID NOs: 1 or 3.Isolated peptidyl portions of ACE-2 proteins can be obtained byscreening peptides recombinantly produced from the correspondingfragment of the nucleic acid encoding such peptides. In addition,fragments can be chemically synthesized using techniques known in theart such as conventional Merrifield solid phase f-Moc or t-Bocchemistry. For example, an ACE-2 polypeptide of the present inventionmay be arbitrarily divided into fragments of desired length with nooverlap of the fragments, or preferably divided into overlappingfragments of a desired length. The fragments can be produced(recombinantly or by chemical synthesis) and tested to identify thosepeptidyl fragments which can function as either agonists or antagonistsof a wild-type (e.g., “authentic”) ACE-2 protein.

An ACE-2 polypeptide can be a membrane bound form or a soluble form. Apreferred soluble ACE-2 polypeptide is a polypeptide which does notcontain the transmembrane domain located from about amino acid 741 toabout amino acid 765 of SEQ ID NO:2. It is likely that there are naturalforms of ACE-2 which fail to contain this domain. Alternatively, suchproteins can be created by genetic engineering by methods known in theart. Soluble ACE-2 proteins can comprise an amino acid sequence fromabout amino acid 19 to about amino acid 740 of SEQ ID NO:2 or homologsthereof. Such proteins can further comprise the amino acid sequence fromabout amino acid 765 to about amino acid 805 of SEQ ID NO:2.Alternatively, soluble ACE-2 proteins can comprise the signal sequence,i.e., amino acids 1-18 of SEQ ID NO:2 or a heterologous signal sequence,which is necessary for obtaining secretion of the protein.

It has been reported membraneous ACE proteins can become detached fromthe membrane to become soluble forms of ACE as the result of apost-translational proteolytic processing event. A metalloproteaseresponsible for this effect has been isolated and referred to as“secretase” (see, e.g., Parvathy et al. (1997) Biochem. J. 327:37).Accordingly, it is likely that ACE-2 proteins are similarly renderedsoluble. Thus, the invention also provides ACE-2 proteins in which thesite of cleavage is modified, such that a secretase would not be able tohydrolyze ACE-2.

In general, polypeptides referred to herein as having an activity (e.g.,are “bioactive”) of an ACE-2 protein are defined as polypeptides whichinclude an amino acid sequence encoded by all or a portion of thenucleic acid sequences shown in one of SEQ ID NOs: 1 or 3 and whichmimic or antagonize all or a portion of the biological/biochemicalactivities of a naturally occurring ACE-2 protein. Examples of suchbiological activity include the ability to catalyze hydrolysis ofangiotensin I into Ang.(1-9), the ability to catalyze hydrolysis of akinin or derivative thereof; the ability to catalyze the hydrolysis ofkinetensin; the ability to bind to a metal ion, e.g., zinc, the abilityto interact with a substrate, e.g., angiotensin I or a kinin, theability to compete with ACE for a target peptide, e.g, angiotensin I;the ability to function as an analgesic, the ability to function as ananti-inflammatory agent the ability to modulate release of histaminefrom mast cells, and the ability to modulate blood cell wallpermeability, blood pressure or vasocontriction. Other biologicalactivities of the subject ACE-2 proteins are described herein or will bereasonably apparent to those skilled in the art. According to thepresent invention, a polypeptide has biological activity if it is aspecific agonist or antagonist of a naturally-occurring form of an ACE-2protein.

A preferred ACE-2 polypeptide having a biological activity is an ACE-2polypeptide comprising a zinc binding domain, e.g, an amino acidsequence from amino acid 374 to amino acid 378 of SEQ ID NO:2. Such azinc binding domain is present in all ACE proteins identified to thisdate and has been identified as being located in the catalytic site ofthe enzyme (Lattion et al. (1989) FEBS Letters 252:99). All the aminoacids which have been reported as interacting with the zinc atom orinvolved in catalysis in ACE proteins are present in ACE-2. Thus, bycomparison, His374, 378 and Glu402 are probably the amino acidscoordinating the zinc atom and Glu375 and His417 are probably involvedin catalysis, as well as Glu 406. Thus, preferred ACE-2 polypeptides canbe generally represented by the formula: X-(ZBD)-Y, wherein ZBDcorresponds to amino acids 374 to 378, 371 to 380, or 372 to 381 and Xand Y represent a certain number of amino acids located upstream anddownstream of ZBD, respectively. X and Y can be any number of aminoacids, including 0, 1, 2, 5, 10, 20, 50, 100, 200, or 300. The ZBD canalso be an extended ZBD, e.g., including the amino acid sequence fromabout amino acid 374 to about amino acid 420 of SEQ ID NO:2 andcontaining all the amino acids likely to be involved in the active siteof the enzyme. ZBD can also be a mutant ZBD, which is, e.g, incapable ofbinding a zinc atom, and thus incapable of catalyzing the hydrolysis ofa peptide. For example, a mutant ZBD can be a ZBD in which His 374, Glu375, His 378, Glu 402, Glu 406, and/or His 417 are replaced by anotheramino acid. Mutated ACE-2 proteins can be used, e.g., as dominantnegative ACE-2 proteins which is capable of interacting with the targetpeptide, but fails to hydrolyze the target peptide and thus competeswith the wild-type ACE-2 or with an ACE protein.

Assays for determining whether a compound, e.g, a protein, such as anACE-2 protein or variant thereof, has one or more of the abovebiological activities are well known in the art. For example, assays fordetermining whether an ACE-2 protein, homolog, or fragment thereof iscapable of catalyzing hydrolysis of a peptide can be performed asdescribed in the Examples. Peptides for use as test substrates can beprepared according to methods known in the art or can be obtainedcommercially.

Other preferred proteins of the invention are those encoded by thenucleic acids set forth in the section pertaining to nucleic acids ofthe invention. In particular, the invention provides fusion proteins,e.g., ACE-2-immunoglobulin fusion proteins. Such fusion proteins canprovide, e.g., enhanced stability and solubility of ACE-2 proteins andmay thus be useful in therapy. Fusion proteins can also be used toproduce an immunogenic fragment of an ACE-2 protein. For example, theVP6 capsid protein of rotavirus can be used as an immunologic carrierprotein for portions of the ACE-2 polypeptide, either in the monomericform or in the form of a viral particle. The nucleic acid sequencescorresponding to the portion of a subject ACE-2 protein to whichantibodies are to be raised can be incorporated into a fusion geneconstruct which includes coding sequences for a late vaccinia virusstructural protein to produce a set of recombinant viruses expressingfusion proteins comprising ACE-2 epitopes as part of the virion. It hasbeen demonstrated with the use of immunogenic fusion proteins utilizingthe Hepatitis B surface antigen fusion proteins that recombinantHepatitis B virions can be utilized in this role as well. Similarly,chimeric constructs coding for fusion proteins containing a portion ofan ACE-2 protein and the poliovirus capsid protein can be created toenhance immunogenicity of the set of polypeptide antigens (see, forexample, EP Publication No: 0259149; and Evans et al. (1989) Nature339:385; Huang et al. (1988) J. Virol. 62:3855; and Schlienger et al.(1992) J. Virol. 66:2).

The Multiple antigen peptide system for peptide-based immunization canalso be utilized to generate an immunogen, wherein a desired portion ofan ACE-2 polypeptide is obtained directly from organo-chemical synthesisof the peptide onto an oligomeric branching lysine core (see, forexample, Posnett et al. (1988) JBC 263:1719 and Nardelli et al. (1992)J. Immunol. 148:914). Antigenic determinants of ACE-2 proteins can alsobe expressed and presented by bacterial cells.

In addition to utilizing fusion proteins to enhance immunogenicity, itis widely appreciated that fusion proteins can also facilitate theexpression of proteins, and accordingly, can be used in the expressionof the ACE-2 polypeptides of the present invention. For example, ACE-2polypeptides can be generated as glutathione-S-transferase (GST-fusion)proteins. Such GST-fusion proteins can enable easy purification of theACE-2 polypeptide, as for example by the use of glutathione-derivatizedmatrices (see, for example, Current Protocols in Molecular Biology, eds.Ausubel et al. (N.Y.: John Wiley & Sons, 1991)).

The present invention further pertains to methods of producing thesubject ACE-2 polypeptides. For example, a host cell transfected with anucleic acid vector directing expression of a nucleotide sequenceencoding the subject polypeptides can be cultured under appropriateconditions to allow expression of the peptide to occur. Suitable mediafor cell culture are well known in the art. The recombinant ACE-2polypeptide can be isolated from cell culture medium, host cells, orboth using techniques known in the art for purifying proteins includingion-exchange chromatography, gel filtration chromatography,ultrafiltration, electrophoresis, and immunoaffinity purification withantibodies specific for such peptide. In a preferred embodiment, therecombinant ACE-2 polypeptide is a fusion protein containing a domainwhich facilitates its purification, such as GST fusion protein.

Moreover, it will be generally appreciated that, under certaincircumstances, it may be advantageous to provide homologs of one of thesubject ACE-2 polypeptides which function in a limited capacity as oneof either an ACE-2 agonist (mimetic) or an ACE-2 antagonist, in order topromote or inhibit only a subset of the biological activities of thenaturally-occurring form of the protein. Thus, specific biologicaleffects can be elicited by treatment with a homolog of limited function,and with fewer side effects relative to treatment with agonists orantagonists which are directed to all of the biological activities ofnaturally occurring forms of ACE-2 proteins.

Homologs of each of the subject ACE-2 proteins can be generated bymutagenesis, such as by discrete point mutation(s), or by truncation.For instance, mutation can give rise to homologs which retainsubstantially the same, or merely a subset, of the biological activityof the ACE-2 polypeptide from which it was derived. Alternatively,antagonistic forms of the protein can be generated which are able toinhibit the function of the naturally occurring form of the protein,such as by competitively binding to an ACE-2 receptor.

The recombinant ACE-2 polypeptides of the present invention also includehomologs of the wildtype ACE-2 proteins, such as versions of thoseproteins which are resistant to proteolytic cleavage, as for example,due to mutations which alter ubiquitination or other enzymatic targetingassociated with the protein.

ACE-2 polypeptides may also be chemically modified to create ACE-2derivatives by forming covalent or aggregate conjugates with otherchemical moieties, such as glycosyl groups, lipids, phosphate, acetylgroups and the like. Covalent derivatives of ACE-2 proteins can beprepared by linking the chemical moieties to functional groups on aminoacid sidechains of the protein or at the N-terminus or at the C-terminusof the polypeptide.

Modification of the structure of the subject ACE-2 polypeptides can befor such purposes as enhancing therapeutic or prophylactic efficacy,stability (e.g., ex vivo shelf life and resistance to proteolyticdegradation), or post-translational modifications (e.g., to alterphosphorylation pattern of protein). Such modified peptides, whendesigned to retain at least one activity of the naturally-occurring formof the protein, or to produce specific antagonists thereof, areconsidered functional equivalents of the ACE-2 polypeptides described inmore detail herein. Such modified peptides can be produced, forinstance, by amino acid substitution, deletion, or addition. Thesubstitutional variant may be a substituted conserved amino acid or asubstituted non-conserved amino acid.

For example, it is reasonable to expect that an isolated replacement ofa leucine with an isoleucine or valine, an aspartate with a glutamate, athreonine with a serine, or a similar replacement of an amino acid witha structurally related amino acid (i.e. isosteric and/or isoelectricmutations) will not have a major effect on the biological activity ofthe resulting molecule. Conservative replacements are those that takeplace within a family of amino acids that are related in their sidechains. Genetically encoded amino acids can be divided into fourfamilies: (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine,histidine; (3) nonpolar=alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar=glycine,asparagine, glutamine, cysteine, serine, threonine, tyrosine. In similarfashion, the amino acid repertoire can be grouped as (1)acidic=aspartate, glutamate; (2) basic=lysine, arginine histidine, (3)aliphatic=glycine, alanine, valine, leucine, isoleucine, serine,threonine, with serine and threonine optionally be grouped separately asaliphatic-hydroxyl; (4) aromatic=phenylalanine, tyrosine, tryptophan;(5) amide=asparagine, glutamine; and (6) sulfur -containing=cysteine andmethionine. (see, for example, Biochemistry, 2^(nd) ed., Ed. by L.Stryer, W H Freeman and Co.: 1981). Whether a change in the amino acidsequence of a peptide results in a functional ACE-2 homolog (e.g.,functional in the sense that the resulting polypeptide mimics orantagonizes the wild-type form) can be readily determined by assessingthe ability of the variant peptide to produce a response in cells in afashion similar to the wild-type protein, or competitively inhibit sucha response. Polypeptides in which more than one replacement has takenplace can readily be tested in the same manner.

This invention further contemplates a method for generating sets ofcombinatorial mutants of the subject ACE-2 proteins as well astruncation mutants, and is especially useful for identifying potentialvariant sequences (e.g., homologs). The purpose of screening suchcombinatorial libraries is to generate, for example, novel ACE-2homologs which can act as either agonists or antagonist, oralternatively, possess novel activities all together. Thus,combinatorially-derived homologs can be generated to have an increasedpotency relative to a naturally occurring form of the protein.

In one embodiment, the variegated library of ACE-2 variants is generatedby combinatorial mutagenesis at the nucleic acid level, and is encodedby a variegated gene library. For instance, a mixture of syntheticoligonucleotides can be enzymatically ligated into gene sequences suchthat the degenerate set of potential ACE-2 sequences are expressible asindividual polypeptides, or alternatively, as a set of larger fusionproteins (e.g., for phage display) containing the set of ACE-2 sequencestherein.

There are many ways by which such libraries of potential ACE-2 homologscan be generated from a degenerate oligonucleotide sequence. Chemicalsynthesis of a degenerate gene sequence can be carried out in anautomatic DNA synthesizer, and the synthetic genes then ligated into anappropriate expression vector. The purpose of a degenerate set of genesis to provide, in one mixture, all of the sequences encoding the desiredset of potential ACE-2 sequences. The synthesis of degenerateoligonucleotides is well known in the art (see for example, Narang, SA(1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc3^(rd) Cleveland Sympos. Macromolecules, ed. A G Walton, Amsterdam:Elsevier pp 273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323;Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic AcidRes. 11:477. Such techniques have been employed in the directedevolution of other proteins (see, for example, Scott et al. (1990)Science 249:386-390; Roberts et al. (1992) PNAS 89:2429-2433; Devlin etal. (1990) Science 249: 404-406; Cwirla et al. (1990) PNAS 87:6378-6382; as well as U.S. Pat. Nos. 5,223,409, 5,198,346, and5,096,815).

Likewise, a library of coding sequence fragments can be provided for anACE-2 clone in order to generate a variegated population of ACE-2fragments for screening and subsequent selection of bioactive fragments.A variety of techniques are known in the art for generating suchlibraries, including chemical synthesis. In one embodiment, a library ofcoding sequence fragments can be generated by (i) treating a doublestranded PCR fragment of an ACE-2 coding sequence with a nuclease underconditions wherein nicking occurs only about once per molecule; (ii)denaturing the double stranded DNA; (iii) renaturing the DNA to formdouble stranded DNA which can include sense/antisense pairs fromdifferent nicked products; (iv) removing single stranded portions fromreformed duplexes by treatment with SI nuclease; and (v) ligating theresulting fragment library into an expression vector. By this exemplarymethod, an expression library can be derived which codes for N-terminal,C-terminal and internal fragments of various sizes.

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations ortruncation, and for screening cDNA libraries for gene products having acertain property. Such techniques will be generally adaptable for rapidscreening of the gene libraries generated by the combinatorialmutagenesis of ACE-2 homologs. The most widely used techniques forscreening large gene libraries typically comprises cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates relatively easy isolation of the vector encodingthe gene whose product was detected. Each of the illustrative assaysdescribed below are amenable to high through-put analysis as necessaryto screen large numbers of degenerate ACE-2 sequences created bycombinatorial mutagenesis techniques.

Combinatorial mutagenesis has a potential to generate very largelibraries of mutant proteins, e.g., in the order of 10²⁶ molecules.Combinatorial libraries of this size may be technically challenging toscreen even with high throughput screening assays. To overcome thisproblem, a new technique has been developed recently, recrusive ensemblemutagenesis (REM), which allows one to avoid the very high proportion ofnon-functional proteins in a random library and simply enhances thefrequency of functional proteins, thus decreasing the complexityrequired to achieve a useful sampling of sequence space. REM is analgorithm which enhances the frequency of functional mutants in alibrary when an appropriate selection or screening method is employed(Arkin and Yourvan, 1992, PNAS USA 89:7811-7815; Yourvan et al., 1992,Parallel Problem Solving from Nature, 2., In Maenner and Manderick,eds., Elsevir Publishing Co., Amsterdam, pp. 401-410; Delgrave et al.,1993, Protein Engineering 6(3):327-331).

The invention also provides for reduction of the ACE-2 proteins togenerate mimetics, e.g., peptide or non-pepide agents, such as smallmolecules, which are able to disrupt binding of an ACE-2 polypeptide ofthe present invention with a molecule, e.g. target peptide. Thus, suchmutagenic techniques as described above are also useful to map thedeterminants of the ACE-2 proteins which participate in protein-proteininteractions involved in, for example, binding of the subject ACE-2polypeptide to a target peptide. To illustrate, the critical residues ofa subject ACE-2 polypeptide which are involved in molecular recognitionof its receptor can be determined and used to generate ACE-2 derivedpeptidomimetics or small molecules which competitively inhibit bindingof the authentic ACE-2 protein with that moiety. By employing, forexample, scanning mutagenesis to map the amino acid residues of thesubject ACE-2 proteins which are involved in binding other proteins,peptidomimetic compounds can be generated which mimic those residues ofthe ACE-2 protein which facilitate the interaction. Such mimetics maythen be used to interfere with the normal function of an ACE-2 protein.For instance, non-hydrolyzable peptide analogs of such residues can begenerated using benzodiazepine (e.g., see Freidinger et al. in Peptides:Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden,Netherlands, 1988), azepine (e.g., see Huffman et al. in Peptides:Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden,Netherlands, 1988), substituted gamma lactam rings (Garvey et al. inPeptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher:Leiden, Netherlands, 1988), keto-methylene pseudopeptides (Ewenson etal. (1986) J Med Chem 29:295; and Ewenson et al. in Peptides: Structureand Function (Proceedings of the 9^(th) American Peptide Symposium)Pierce Chemical Co. Rockland, Ill., 1985), b-turn dipeptide cores (Nagaiet al. (1985) Tetrahedron Lett 26:647; and Sato et al. (1986) J Chem SocPerkin Trans 1:1231), and b-aminoalcohols (Gordon et al. (1985) BiochemBiophys Res Commun126:419; and Dann et al. (1986) Biochem Biophys ResCommun 134:71).

Also within the scope of the invention are kinetensin conversionproducts, such as ACE-2 kinetensin conversion products. A preferredconversion product is kinetensin (1-8) (SEQ ID NO:24). Analogs of suchconversion products are also within the scope of the invention and canbe, e.g., peptidomimetics.

5. Anti-ACE-2 Antibodies and Uses Therefor

Another aspect of the invention pertains to an antibody specificallyreactive with a mammalian ACE-2 protein, e.g., a wild-type or mutatedACE-2 protein. For example, by using immunogens derived from an ACE-2protein, e.g., based on the cDNA sequences, anti-protein/anti-peptideantisera or monoclonal antibodies can be made by standard protocols(See, for example, Antibodies: A Laboratory Manual ed. by Harlow andLane (Cold Spring Harbor Press: 1988)). A mammal, such as a mouse, ahamster or rabbit can be immunized with an immunogenic form of thepeptide (e.g., a mammalian ACE-2 polypeptide or an antigenic fragmentwhich is capable of eliciting an antibody response, or a fusion proteinas described above). Techniques for conferring immunogenicity on aprotein or peptide include conjugation to carriers or other techniqueswell known in the art. An immunogenic portion of an ACE-2 protein can beadministered in the presence of adjuvant. The progress of immunizationcan be monitored by detection of antibody titers in plasma or serum.Standard ELISA or other immunoassays can be used with the immunogen asantigen to assess the levels of antibodies. In a preferred embodiment,the subject antibodies are immunospecific for antigenic determinants ofan ACE-2 protein of a mammal, e.g., antigenic determinants of a proteinset forth in SEQ ID No: 2 or closely related homologs (e.g., at least90% homologous, and more preferably at least 94% homologous). Exemplaryantibodies were obtained and are set forth in the Examples.

Following immunization of an animal with an antigenic preparation of anACE-2 polypeptide, anti-ACE-2 antisera can be obtained and, if desired,polyclonal anti-ACE-2 antibodies isolated from the serum. To producemonoclonal antibodies, antibody-producing cells (lymphocytes) can beharvested from an immunized animal and fused by standard somatic cellfusion procedures with immortalizing cells such as myeloma cells toyield hybridoma cells. Such techniques are well known in the art, andinclude, for example, the hybridoma technique (originally developed byKohler and Milstein, (1975) Nature, 256: 495-497), the human B cellhybridoma technique (Kozbar et al., (1983) Immunology Today, 4: 72), andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc. pp. 77-96). Hybridoma cells can be screened immunochemically forproduction of antibodies specifically reactive with a mammalian ACE-2polypeptide of the present invention and monoclonal antibodies isolatedfrom a culture comprising such hybridoma cells. In one embodimentanti-human ACE-2 antibodies specifically react with the protein encodedby a nucleic acid having SEQ ID NO:1.

The term antibody as used herein is intended to include fragmentsthereof which are also specifically reactive with one of the subjectmammalian ACE-2 polypeptides. Antibodies can be fragmented usingconventional techniques and the fragments screened for utility in thesame manner as described above for whole antibodies. For example, F(ab)₂fragments can be generated by treating antibody with pepsin. Theresulting F(ab)₂ fragment can be treated to reduce disulfide bridges toproduce Fab fragments. The antibody of the present invention is flurtherintended to include bispecific, single-chain, and chimeric and humanizedmolecules having affinity for an ACE-2 protein conferred by at least oneCDR region of the antibody. In preferred embodiments, the antibodies,the antibody further comprises a label attached thereto and able to bedetected, (e.g., the label can be a radioisotope, fluorescent compound,enzyme or enzyme co-factor).

Anti-ACE-2 antibodies can be used, e.g., to monitor ACE-2 protein levelsin an individual for determining, e.g., whether a subject has a diseaseor condition associated with an aberrant ACE-2 protein level, orallowing determination of the efficacy of a given treatment regimen foran individual afflicted with such a disorder. The level of ACE-2polypeptides may be measured from cells and/or in bodily fluid, such asin blood samples saliva, urine, and sweat.

Another application of anti-ACE-2 antibodies of the present invention isin the immunological screening of cDNA libraries constructed inexpression vectors such as λgt11, λgt18-23, λZAP, and λORF8. Messengerlibraries of this type, having coding sequences inserted in the correctreading frame and orientation, can produce fusion proteins. Forinstance, λgt11 will produce fusion proteins whose amino termini consistof β-galactosidase amino acid sequences and whose carboxy terminiconsist of a foreign polypeptide. Antigenic epitopes of an ACE-2protein, e.g., other orthologs of a particular ACE-2 protein or otherparalogs from the same species, can then be detected with antibodies,as, for example, reacting nitrocellulose filters lifted from infectedplates with anti-ACE-2 antibodies. Positive phage detected by this assaycan then be isolated from the infected plate. Thus, the presence ofACE-2 homologs can be detected and cloned from other animals, as canalternate isoforms (including splice variants) from humans.

Antibodies specifically binding to an ACE-2 conversion product are alsowithin the scope of the invention. In an illustrative embodiment, theinvention provides antibodies which specifically bind to kinetensin(1-8) and not to kinetensin (1-9).

6. Transgenic Animals

The invention further provides for transgenic animals, which can be usedfor a variety of purposes, e.g., to identify ACE-2 therapeutics.Transgenic animals of the invention include non-human animals containinga heterologous ACE-2 gene or fragment thereof under the control of anACE-2 promoter or under the control of a heterologous promoter.Accordingly, the transgenic animals of the invention can be animalsexpressing a transgene encoding a wild-type ACE-2 protein or fragmentthereof or variants thereof, including mutants and polymorphic variantsthereof. Such animals can be used, e.g., to determine the effect of adifference in amino acid sequence of an ACE-2 protein from the sequenceset forth in SEQ ID NO:2, such as a polymorphic difference. Theseanimals can also be used to determine the effect of expression of anACE-2 protein in a specific site or for identifying ACE-2 therapeuticsor confirming their activity in vivo. Exemplary transgenic mice aredescribed in the Examples.

The transgenic animals can also be animals containing a transgene, suchas reporter gene, under the control of an ACE-2 promoter or fragmentthereof. These animals are useful, e.g., for identifying ACE-2 drugsthat modulate production of ACE-2, such as by modulating ACE-2 geneexpression. An ACE-2 gene promoter can be isolated, e.g., by screeningof a genomic library with an ACE-2 cDNA fragment and characterizedaccording to methods known in the art.

Yet other non-human animals within the scope of the invention includethose in which the expression of the endogenous ACE-2 gene has beenmutated or “knocked out”. These animals could be useful to determinewhether the absence of ACE-2 will result in a specific phenotype, inparticular whether these mice have or are likely to develop a specificdisease, such as high susceptibility to inflammatory reactions. Theseanimals are also useful for determining the effect of a specific aminoacid difference in an ACE-2 gene. In fact these knock out animals can becrossed with transgenic animals expressing, e.g., a mutated form ofACE-2, thus resulting in an animal which expresses only the mutatedprotein and not the wild-type ACE-2 protein. Exemplary knock out miceare described in the Examples.

Methods for obtaining transgenic and knockout non-human animals are wellknown in the art.

7. Screening Assays for ACE-2 Therapeutics

The invention further provides screening methods for identifying ACE-2therapeutics, e.g., for treating diseases or conditions caused by, orcontributed to by an abnormal ACE-2 activity or which can benefit from amodulation of an ACE-2 activity or protein level, e.g., hypertension,hypotension, arrhythmia, or CHF. An ACE-2 therapeutic can be any type ofcompound, including a protein, a peptide, peptidomimetic, smallmolecule, and nucleic acid. A nucleic acid can be, e.g., a gene, anantisense nucleic acid, a ribozyme, or a triplex molecule. An ACE-2therapeutic of the invention can be an agonist or an antagonist.Preferred ACE-2 agonists include ACE-2 proteins or derivatives thereofwhich mimic at least one ACE-2 activity, e.g., the capability tocatalyze hydrolysis of a target peptide or nucleic acids encoding such.Other preferred agonists include compounds which are capable ofincreasing the production of an ACE-2 protein in a cell, e.g., compoundscapable of upregulating the expression of an ACE-2 gene, and compoundswhich are capable of enhancing an ACE-2 activity and/or the interactionof an ACE-2 protein with another molecule, such as a target peptide.Preferred ACE-2 antagonists include ACE-2 proteins which are dominantnegative proteins, which, e.g., are capable of binding to, but not tohydrolyze target peptides. Other preferred antagonists include compoundswhich decrease or inhibit the production of an ACE-2 protein in a celland compounds which are capable of downregulating expression of an ACE-2gene, and compounds which are capable of donwregulating an ACE-2activity and/or interaction of an ACE-2 protein with another molecule,such as a target peptide, e.g, angiotensin I, a kinin, kinetensin, orneurotensin. In another preferred embodiment, an ACE-2 antagonist is amodified form of a target peptide, which is capable of interacting withthe catalytic site of an ACE-2 protein, but which does not havebiological activity, e.g., which is not vasopressive.

The invention also provides screening methods for identifying ACE-2therapeutics which are capable of binding to an ACE-2 protein, e.g., awild-type ACE-2 protein or a mutated form of an ACE-2 protein, andthereby modulate the catalytic activity of the ACE-protein or degradesor causes the ACE-2 protein to be degraded. For example, such an ACE-2therapeutic can be an antibody or derivative thereof which interactsspecifically with an ACE-2 protein (either wild-type or mutated).

Thus, the invention provides screening methods for identifying ACE-2agonist and antagonist compounds, comprising selecting compounds whichare capable of interacting with an ACE-2 protein or with a moleculeinteracting with an ACE-2 protein such as a target peptide and/orcompounds which are capable of modulating the interaction of an ACE-2protein with another molecule, such as a target peptide. In general, amolecule which is capable of interacting with an ACE-2 protein isreferred to herein as “ACE-2 binding partner” and can be a targetpeptide, e.g., angiotensin I, a kinin, kinetensin, or neurotensin or ananalog thereof or a portion thereof, so long as the analog or portion ofthe target peptide is capable of binding to an ACE-2 polyptide andoptionally of being cleaved by an ACE-2 polypeptide. An ACE-2 bindingpartner can also be a polypeptide which is not a target peptide andwhich may, e.g., interact with an ACE-2 protein at sites other than thecatalytic site.

The compounds of the invention can be identified using various assaysdepending on the type of compound and activity of the compound that isdesired. Set forth below are at least some assays that can be used foridentifying ACE-2 therapeutics. It is within the skill of the art todesign additional assays for identifying ACE-2 therapeutics.

7.1 Cell-Free Assays

Cell-free assays can be used to identify compounds which are capable ofinteracting with an ACE-2 protein or binding partner, to thereby modifythe activity of the ACE-2 protein or binding partner. Such a compoundcan, e.g., modify the structure of an ACE-2 protein or binding partnerand thereby effect its activity. Cell-free assays can also be used toidentify compounds which modulate the interaction between an ACE-2protein and an ACE-2 binding partner, such as a target peptide. In apreferred embodiment, cell-free assays for identifying such compoundsconsist essentially in a reaction mixture containing an ACE-2 proteinand a test compound or a library of test compounds in the presence orabsence of a binding partner. A preferred binding partner is angiotensinI or kinetensin or portions thereof sufficient for interacting withACE-2. A test compound can be, e.g., a derivative of an ACE-2 bindingpartner, e.g., an biologically inactive target peptide, or a smallmolecule.

Accordingly, one exemplary screening assay of the present inventionincludes the steps of contacting an ACE-2 protein or functional fragmentthereof or an ACE-2 binding partner with a test compound or library oftest compounds and detecting the formation of complexes. For detectionpurposes, the molecule can be labeled with a specific marker and thetest compound or library of test compounds labeled with a differentmarker. Interaction of a test compound with an Al:Z protein or fragmentthereof or ACE-2 binding partner can then be detected by determining thelevel of the two labels after an incubation step and a washing step. Thepresence of two labels after the washing step is indicative of aninteraction.

An interaction between molecules can also be identified by usingreal-time BIA (Biomolecular Interaction Analysis, Pharmacia BiosensorAB) which detects surface plasmon resonance (SPR), an opticalphenomenon. Detection depends on changes in the mass concentration ofmacromolecules at the biospecific interface, and does not require anylabeling of interactants. In one embodiment, a library of test compoundscan be immobilized on a sensor surface, e.g., which forms one wall of amicro-flow cell. A solution containing the ACE-2 protein, functionalfragment thereof, ACE-2 analog or ACE-2 binding partner is then flowncontinuously over the sensor surface. A change in the resonance angle asshown on a signal recording, indicates that an interaction has occurred.This technique is further described, e.g., in BIAtechnology Handbook byPharmacia.

Another exemplary screening assay of the present invention includes thesteps of (a) forming a reaction mixture including: (i) an ACE-2polypeptide, (ii) an ACE-2 binding partner (e.g., angiotensin I, akinin, or kinetensin), and (iii) a test compound; and (b) detectinginteraction of the ACE-2 and the ACE-2 binding protein. The ACE-2polypeptide and ACE-2 binding partner can be produced recombinantly,purified from a source, e.g., plasma, or chemically synthesized, asdescribed herein. A statistically significant change (potentiation orinhibition) in the interaction of the ACE-2 and ACE-2 binding protein inthe presence of the test compound, relative to the interaction in theabsence of the test compound, indicates a potential agonist (mimetic orpotentiator) or antagonist (inhibitor) of ACE-2 bioactivity for the testcompound. The compounds of this assay can be contacted simultaneously.Alternatively, an ACE-2 protein can first be contacted with a testcompound for an appropriate amount of time, following which the ACE-2binding partner is added to the reaction mixture. The efficacy of thecompound can be assessed by generating dose response curves from dataobtained using various concentrations of the test compound. Moreover, acontrol assay can also be performed to provide a baseline forcomparison. In the control assay, isolated and purified ACE-2polypeptide or binding partner is added to a composition containing theACE-2 binding partner or ACE-2 polypeptide, and the formation of acomplex is quantitated in the absence of the test compound.

Complex formation between an ACE-2 protein and an ACE-2 binding partnermay be detected by a variety of techniques. Modulation of the formationof complexs can be quantitated using, for example, detectably labeledproteins such as radiolabeled, fluorescently labeled, or enzymaticallylabeled ACE-2 proteins or ACE-2 binding partners, by immunoassay, or bychromatographic detection.

Typically, it will be desirable to immobilize either ACE-2 or itsbinding partner to facilitate separation of complexes from uncomplexedforms of one or both of the proteins, as well as to accommodateautomation of the assay. Binding of ACE-2 to an ACE-2 binding partner,can be accomplished in any vessel suitable for containing the reactants.Examples include microtitre plates, test tubes, and micro-centrifugetubes. In one embodiment, a fusion protein can be provided which adds adomain that allows the protein to be bound to a matrix. For example,glutathione-S-transferase/ACE-2 (GST/ACE-2) fusion proteins can beadsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized microtitre plates, which are thencombined with the ACE-2 binding partner, e.g. an ³⁵S-labeled ACE-2binding partner, and the test compound, and the mixture incubated underconditions conducive to complex formation, e.g. at physiologicalconditions for salt and pH, though slightly more stringent conditionsmay be desired. Following incubation, the beads are washed to remove anyunbound label, and the matrix immobilized and radiolabel determineddirectly (e.g. beads placed in scintilant), or in the supernatant afterthe complexes are subsequently dissociated. Alternatively, the complexescan be dissociated from the matrix, separated by SDS-PAGE, and the levelof ACE-2 protein or ACE-2 binding partner found in the bead fractionquantitated from the gel using standard electrophoretic techniques suchas described in the appended examples.

Other techniques for immobilizing proteins on matrices are alsoavailable for use in the subject assay. For instance, either ACE-2 orits cognate binding partner can be immobilized utilizing conjugation ofbiotin and streptavidin. For instance, biotinylated ACE-2 molecules canbe prepared from biotin-NHS (N-hydroxy-succinimide) using techniqueswell known in the art (e.g., biotinylation kit, Pierce Chemicals,Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96well plates (Pierce Chemical). Alternatively, antibodies reactive withACE-2 can be derivatized to the wells of the plate, and ACE-2 trapped inthe wells by antibody conjugation. As above, preparations of an ACE-2binding protein and a test compound are incubated in the ACE-2presenting wells of the plate, and the amount of complex trapped in thewell can be quantitated. Exemplary methods for detecting such complexes,in addition to those described above for the GST-immobilized complexes,include immunodetection of complexes using antibodies reactive with theACE-2 binding partner, or which are reactive with ACE-2 protein andcompete with the binding partner; as well as enzyme-linked assays whichrely detecting an enzymatic activity associated with the bindingpartner, either intrinsic or extrinsic activity. In the instance of thelatter, the enzyme can be chemically conjugated or provided as a fusionprotein with the ACE-2 binding partner. To illustrate, the ACE-2 bindingpartner can be chemically cross-linked or genetically fused withhorseradish peroxidase, and the amount of polypeptide trapped in thecomplex can be assessed with a chromogenic substrate of the enzyme, e.g.3,3′-diamino-benzadine terahydrochloride or 4-chloro-1-napthol.Likewise, a fusion protein comprising the polypeptide andglutathione-S-transferase can be provided, and complex formationquantitated by detecting the GST activity using1-chloro-2,4-dinitrobenzene (Habig et al (1974) J Biol Chem 249:7130).

For processes which rely on immunodetection for quantitating one of theproteins trapped in the complex, antibodies against the protein, such asanti-ACE-2 antibodies, can be used. Alternatively, the protein to bedetected in the complex can be “epitope tagged” in the form of a fusionprotein which includes, in addition to the ACE-2 sequence, a secondpolypeptide for which antibodies are readily available (e.g. fromcommercial sources). For instance, the GST fusion proteins describedabove can also be used for quantification of binding using antibodiesagainst the GST moiety. Other useful epitope tags include myc-epitopes(e.g., see Ellison et al. (1991) J Biol Chem 266:21150-21157) whichincludes a 10-residue sequence from c-myc, as well as the pFLAG system(International Biotechnologies, Inc.) or the pEZZ-protein A system(Pharmacia, N.J.).

Cell-free assays can also be used to identify compounds which interactwith an ACE-2 protein and modulate an activity of an ACE-2 protein.Accordingly, in one embodiment, an ACE-2 protein is contacted with atest compound and the catalytic activity of ACE-2 is monitored. In oneembodiment, the abililty of ACE-2 to bind to and/or to hydrolyze atarget peptide, e.g, angiotensin I, a kinin, such as bradykinin, orkinetensin, is determined. The binding affinity of ACE-2 to a targetpeptide can be determined according to methods known in the art.Determination of the enzymatic activity of ACE-2 can be performed asfurther described herein. In particular, enzymatic activity can bedemonstrated by subjecting a reaction mixture containing an ACE-2 enzymeand a target peptide after incubation, to mass spectometry, as describedin the Examples.

In a preferred embodiment, the invention provides a screening methodwhich comprises combining an ACE-2 polyptide and a target peptidetogether with a test compound in a reaction mixture in conditionssufficient for the ACE-2 polypeptide to cleave the target peptide in theabsence of the test compound. The method further comprises monitoringthe presence of the target peptide, the target peptide conversionproduct, and/or of the one or more amino acids cleaved from the targetpeptide, such that a difference in the amount of at least one of thetarget peptide, the target peptide conversion product or one or moreamino acids in the reaction mixture incubated with the test compoundrelative to a reaction mixture that does not contain the test compoundindicates that the test compound is an ACE-2 therapeutic. In an evenmore preferred embodiment of the invention, the presence and/or theamount of the target peptide, the target peptide conversion product,and/or of the one or more amino acids in the reaction mixture isdetermined by spectrometric analysis of the reaction mixture or of apart thereof., e.g., as described in the Examples.

Assays can also be developed for identifying compounds which interactand optionally inhibit (i) ACE-2, but not ACE; (ii) ACE, but not ACE-2;and (iii) ACE-2 and ACE. This can be done by a two step screening assay,wherein each step can be conducted as described herein.

7.2. Cell Based Assays

In addition to cell-free assays, such as described above, ACE-2 proteinsas provided by the present invention, facilitate the generation ofcell-based assays, e.g., for identifying small molecule agonists orantagonists. In one embodiment, a cell expressing an ACE-2 protein onthe outer surface of its cellular membrane is incubated in the presenceof a test compound alone or a test compound and a molecule which isknown to interact with ACE-2 and the interaction between ACE-2 and atest compound is detected, e.g., by using a microphysiometer (McConnellet al. (1992) Science 257:1906). An interaction between the ACE-2protein the test compound is detected by the microphysiometer as achange in the acidification of the medium.

Cell based assays can also be used to identify compounds which modulateexpression of an ACE-2 gene, modulate translation of an ACE-2 mRNA, orwhich modulate the stability of an ACE-2 mRNA or protein. Accordingly,in one embodiment, a cell which is capable of producing ACE-2, e.g., akidney cell, is incubated with a test compound and the amount of ACE-2produced in the cell medium is measured and compared to that producedfrom a cell which has not been contacted with the test compound. Thespecificity of the compound vis a vis ACE-2 can be confirmed by variouscontrol analysis, e.g., measuring the expression of one or more controlgenes.

Compounds which can be tested include small molecules, proteins, andnucleic acids. In particular, this assay can be used to determine theefficacity of ACE-2 antisense molecules or ribozymes.

In another embodiment, the effect of a test compound on transcription ofan ACE-2 gene is determined by transfection experiments using a reportergene operatively linked to at least a portion of the promoter of anACE-2 gene. A promoter region of a gene can be isolated, e.g., from agenomic library according to methods known in the art. The reporter genecan be any gene encoding a protein which is readily quantifiable, e.g,the luciferase or CAT gene, well known in the art.

This invention further pertains to novel agents identified by theabove-described screening assays and uses thereof for treatments asdescribed herein.

8. Predictive Medicine

The invention further features predictive medicines, which are based, atleast in part, on the identity of the novel ACE-2 genes and alterationsin the genes and related pathway genes, which affect the expressionlevel and/or function of the encoded ACE-2 protein in a subject.

For example, information obtained using the diagnostic assays(Diagnomics™ molecular diagnostics) described herein (alone or inconjunction with information on another genetic defect, whichcontributes to the same disease) is useful for diagnosing or confirmingthat a symptomatic subject (e.g. a subject symptomatic for hypertension,hypotension, CHF, or a kinetensin associated condition), has a geneticdefect (e.g. in an ACE-2 gene or in a gene that regulates the expressionof an ACE-2 gene), which causes or contributes to the particular diseaseor disorder. Alternatively, the information (alone or in conjunctionwith information on another genetic defect, which contributes to thesame disease) can be used prognostically for predicting whether anon-symptomatic subject is likely to develop a disease or condition,which is caused by or contributed to by an abnormal ACE-2 activity orprotein level (e.g. hypertension, hypotension, CHF, or a kinetensinassociated condition) in a subject. For example, the amount of ACE-2protein in a bodily fluid, e.g., blood, serum, saliva or urine, can bedetermined and compared to levels found in individuals that are notknown to suffer from any ACE-2 associated diseases. Based on theprognostic information, a doctor can recommend a regimen (e.g. diet orexercise) or therapeutic protocol, useful for preventing or prolongingonset of the particular disease or condition in the individual.

In addition, knowledge of the particular alteration or alterations,resulting in defective or deficient ACE-2 genes or proteins in anindividual (the ACE-2 genetic profile), alone or in conjunction withinformation on other genetic defects contributing to the same disease(the genetic profile of the particular disease) allows customization oftherapy for a particular disease to the individual's genetic profile,the goal of “pharmacogenomics”. For example, an individual's ACE-2genetic profile or the genetic profile of a disease or condition, towhich ACE-2 genetic alterations cause or contribute, can enable adoctor: 1) to more effectively prescribe a drug that will address themolecular basis of the disease or condition; and 2) to better determinethe appropriate dosage of a particular drug. For example, the expressionlevel of ACE-2 proteins, alone or in conjunction with the expressionlevel of other genes, known to contribute to the same disease, can bemeasured in many patients at various stages of the disease to generate atranscriptional or expression profile of the disease. Expressionpatterns of individual patients can then be compared to the expressionprofile of the disease to determine the appropriate drug and dose toadminister to the patient.

The ability to target populations expected to show the highest clinicalbenefit, based on the ACE-2 or disease genetic profile, can enable: 1)the repositioning of marketed drugs with disappointing market results;2) the rescue of drug candidates whose clinical development has beendiscontinued as a result of safety or efficacy limitations, which arepatient subgroup-specific; and 3) an accelerated and less costlydevelopment for drug candidates and more optimal drug labeling (e.g.since the use of ACE-2 as a marker is useful for optimizing effectivedose).

These and other methods are described in further detail in the followingsections.

8.1. Prognostic and Diagnostic Assays

The present methods provide means for determining if a subject has(diagnostic) or is at risk of developing (prognostic) a disease,condition or disorder that is associated with an aberrant ACE-2activity, e.g., an aberrant level of ACE-2 protein or an aberrantbioactivity. As set forth below, diseases or conditions that can becaused by or contributed to an abnormal ACE-2 level or bioactivityinclude diseases or conditions, which are caused by or contributed to byan abnormal amount of a target peptide of ACE-2 or level of angiotensinor kinetensin conversion product, resulting, e.g., from inappropriatehydrolysis. For example, the target peptide can be angiotensin I and thedisease or condition associated with an aberrant level of an angiotensinconversion product, e.g., angiotensin II level (in this case abnormallyhigh level) can be hypertension or congestive heart failure. In fact, inat least some tissues, an abnormally high angiotensin II level couldresult from an abnormally low activity of an ACE-2 enzyme, therebyallowing most or all of the angiotensin I to be converted intoangiotensin II, instead of Ang.(1-9). Diseases associated with anabnormal kinetensin or kinetensin conversion product include histamineassociated conditions such as allergies. The target peptide can also bea kinin, and the diseases or conditions associated with an aberrantkinin level (in this case an abnormally high kinin level) includeinflammatory diseases, and pain. Furthermore, an abnormally high ACE-2mRNA level has been found in cardiac tissue from individuals which hadCHF (see Examples). Similarly, ACE has been found to be present atincreased levels in the free wall, septum and apex of the hypertrophiedleft ventricle (Schunkert et al. (1990) J. Clin. Invest. 86:1913). Thus,it is believed that an abnormally high level of ACE-2 correlates withheart failure. Accordingly, the invention provides methods fordetermining whether a subject has or is likely to develop hypertension,hypotension, congestive heart failure, or a kinetensin associatedcondition, for example, comprising determining the level of an ACE-2gene or protein, an ACE-2 bioactivity and/or the presence of a mutationor particular polymorphic variant in the ACE-2 gene. The invention alsoprovides methods for determining whether the pain of a subject is causedby an abnormally low level of ACE-2.

Since ACE-2 catalyzes hydrolysis of yet other peptides, e.g,neuropeptides such as neurotensin, the invention also provides methodsfor diagnosing, for example, neurological diseases or other diseasescaused by inappropriate levels of ACE-2 target peptides, or conversionproducts thereof. Thus, ACE-2 therapeutics may be used for the treatmentof neuropsychiatric disorders, especially those associated with adysfumction of the dopaminergic systems, for example psychoses, moreespecially schizophrenia, and diseases of movement such as Parkinson'sdisease (D. R. Handrich et al., Brain Research, 1982, 231, 216-221 andC. B. Nemeroff, Biological Psychiatry, 1980, 15 (2), 283-302). They maybe used to diagnose and/or treat malignant neoplastic diseases, forexample human meningiomas which are not surgically accessible (P.Mailleux, Peptides, 1990, 11, 1245-1253), cancers of the prostate (I.Sehgal et al., Proc. Nat. Acad. Sci., 1994, 91, 4673-4677) and smallcell cancers of the lung (T. Sethi et al., Cancer Res., 1991, 51,3621-3623). They may be used in the treatment of motor, secretory,ulcerous and/or tumoral gastrointestinal disorders (review by A. Shulkesin “Gut Peptides: Biochemistry and Physiology, Ed. J. Waish and G. J.Dockray, 1994”). ACE-2 therapeutics can be used in the treatment ofcomplaints such as: irritable bowel syndrome, diarrhoea, colitis,ulcers, tumours of the gastrointestinal tract, dyspepsia, pancreatitisand oesophagitis. They may also be of value as modulators of food intake(Beck, B. Metabolism, 1995, 44, 972-975). The compounds according to theinvention may be indicated as diuretics, and for treating certaindisorders caused by stress, such as migraines, neurogenic pruritus andinterstitial cystitis (Theoharides T. C. et al., Endocrinol., 1995, 136,5745-5750). The compounds of the present invention may also be of valuein analgesia, by acting on the effects of morphine (M. O. Urban, J.Pharm. Exp. Ther., 1993, 265, 2, 580-586).

Since ACE-2 also appears to be involved in disorders associated withabnormalities in the conduction system of the heart, such asarrhythmias, the present invention also provides methods for determiningif a subject has or is at risk of developing a disorder associated withabnormalities in the conduction system of the heart, such as anarrhythmia.

In one embodiment, the method comprises determining whether a subjecthas an abnormal mRNA and/or protein level of ACE-2, such as by Northernblot analysis, reverse transcription-polymerase chain reaction (RT-PCR),in situ hybridization, immunoprecipitation, Western blot hybridization,or immunohistochemistry. According to the method, cells are obtainedfrom a subject and the ACE-2 protein or mRNA level is determined andcompared to the level of ACE-2 protein or mRNA level in a healthysubject. An abnormal level of ACE-2 polypeptide or mRNA level is likelyto be indicative of an aberrant ACE-2 activity.

In another embodiment, the method comprises measuring at least oneactivity of ACE-2. For example, the catalytic activity of ACE-2, e.g.,capability to catalyze hydrolysis of certain peptides, e.g., angiotensinand/or kinins, can be determined, e.g., as described herein. Similarly,the constant of affinity of an ACE-2 protein of a subject with a targetpeptide, e.g. angiotensin or a kinin, can be determined. Comparison ofthe results obtained with results from similar analysis performed onACE-2 proteins from healthy subjects will be indicative of whether asubject has an abnormal ACE-2 activity. Measurement of Ang.(1-9),Ang.(1-5), and/or kinetensin (1-8), level can also be used as anindicator of the activity of an ACE-2 enzyme in a subject. Suchmeasurements can be done by spectrometry, as described herein.

In preferred embodiments, the methods for determining whether a subjecthas or is at risk for developing a disease associated with an aberrantACE-2 activity is characterized as comprising detecting, in a sample ofcells from the subject, the presence or absence of a genetic alterationcharacterized by at least one of (i) an alteration affecting theintegrity of a gene encoding an ACE-2 polypeptide, or (ii) themis-expression of the ACE-2 gene. To illustrate, such geneticalterations can be detected by ascertaining the existence of at leastone of (i) a deletion of one or more nucleotides from an ACE-2 gene,(ii) an addition of one or more nucleotides to an ACE-2 gene, (iii) asubstitution of one or more nucleotides of an ACE-2 gene, (iv) a grosschromosomal rearrangement of an ACE-2 gene, (v) a gross alteration inthe level of a messenger RNA transcript of an ACE-2 gene, (vii) aberrantmodification of an ACE-2 gene, such as of the methylation pattern of thegenomic DNA, (vii) the presence of a non-wild type splicing pattern of amessenger RNA transcript of an ACE-2 gene, (viii) a non-wild type levelof an ACE-2 polypeptide, (ix) allelic loss of an ACE-2 gene, and/or (x)inappropriate post-translational modification of an ACE-2 polypeptide.As set out below, the present invention provides a large number of assaytechniques for detecting alterations in an ACE-2 gene. These methodsinclude, but are not limited to, methods involving sequence analysis,Southern blot hybridization, restriction enzyme site mapping, andmethods involving detection of absence of nucleotide pairing between thenucleic acid to be analyzed and a probe. These and other methods arefurther described infra.

Specific diseases or disorders, e.g., genetic diseases or disorders, areassociated with specific allelic variants of polymorphic regions ofcertain genes, which do not necessarily encode a mutated protein. Thus,the presence of a specific allelic variant of a polymorphic region of agene, such as a single nucleotide polymorphism (“SNP”), in a subject canrender the subject susceptible to developing a specific disease ordisorder. Polymorphic regions have been identified in the human ACE-2gene (see Examples). The link with a specific disease can now readily bedetermined by studying specific populations of individuals, e.g,individuals which developed a specific disease, such as hypertension orCHF. A polymorphic region can be located in any region of a gene, e.g.,exons, in coding or non coding regions of exons, introns, and promoterregion.

Allelic variants of the human ACE gene have also been described and insome cases allelic variants have been found to be strongly associatedwith a higher risk for acute coronary events, sudden cardiac death,vascular restenosis after angioplasty, and idiopathic and hypertrophiccardiomyopathy (Malik et al. (1997) Am Heart J. 134:514). Thus, theinvention provides methods for determining the identity of the allele orallelic variant of a polymorphic region of an ACE-2 gene, and optionallyACE gene, in a subject, to thereby determine whether the subject has oris at risk of developing a disease or disorder associated with aspecific allelic variant of a polymorphic region.

In an exemplary embodiment, there is provided a nucleic acid compositioncomprising a nucleic acid probe including a region of nucleotidesequence which is capable of hybridizing to a sense or antisensesequence of an ACE-2 gene or naturally occurring mutants thereof, or 5′or 3′ flanking sequences or intronic sequences naturally associated withthe subject ACE-2 genes or naturally occurring mutants thereof. Thenucleic acid of a cell is rendered accessible for hybridization, theprobe is contacted with the nucleic acid of the sample, and thehybridization of the probe to the sample nucleic acid is detected. Suchtechniques can be used to detect alterations or allelic variants ateither the genomic or mRNA level, including deletions, substitutions,etc., as well as to determine mRNA transcript levels.

A preferred detection method is allele specific hybridization usingprobes overlapping the mutation or polymorphic site and having about 5,10, 20, 25, or 30 nucleotides around the mutation or polymorphic region,e.g., probes comprising or hybridizing specifically to SEQ ID NOs:87,89, 91, 93, 95, 97, 99, and 101. Preferably these probes do nothybridize to SEQ ID NOs:86, 88, 90, 92, 94, 96, 98, or 100 or thecomplements thereof. Other preferred probes hybridize specifically toSEQ ID NOs: 86, 88, 90, 92, 94, 96, 98, or 100 or complement thereof,but not to SEQ ID NOs: 87, 89, 91, 93, 95, 97, 99, and 101 or complementthereof. In a preferred embodiment of the invention, several probescapable of hybridizing specifically to allelic variants, such as singlenucleotide polymorphisms, are attached to a solid phase support, e.g., a“chip”. Oligonucleotides can be bound to a solid support by a variety ofprocesses, including lithography. For example a chip can hold up to250,000 oligonucleotides. Mutation detection analysis using these chipscomprising oligonucleotides, also termed “DNA probe arrays” is describede.g., in Cronin et al. (1996) Human Mutation 7:244. In one embodiment, achip comprises all the allelic variants of at least one polymorphicregion of a gene. The solid phase support is then contacted with a testnucleic acid and hybridization to the specific probes is detected.Accordingly, the identity of numerous allelic variants of one or moregenes can be identified in a simple hybridization experiment.

In certain embodiments, detection of the alteration comprises utilizingthe probe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S.Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligase chain reaction (LCR) (see, e.g., Landegran etal. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) PNAS91:360-364), the latter of which can be particularly useful fordetecting point mutations in the ACE-2 gene (see Abravaya et al. (1995)Nuc Acid Res 23:675-682). In a merely illustrative embodiment, themethod includes the steps of (i) collecting a sample of cells from apatient, (ii) isolating nucleic acid (e.g., genomic, mRNA or both) fromthe cells of the sample, (iii) contacting the nucleic acid sample withone or more primers which specifically hybridize to an ACE-2 gene underconditions such that hybridization and amplification of the ACE-2 gene(if present) occurs, and (iv) detecting the presence or absence of anamplification product, or detecting the size of the amplificationproduct and comparing the length to a control sample. It is anticipatedthat PCR and/or LCR may be desirable to use as a preliminaryamplification step in conjunction with any of the techniques used fordetecting mutations described herein. Preferred hybridization primersare set forth in the Examples.

Alternative amplification methods include: self sustained sequencereplication (Guatelli, J. C. et al., 1990, Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh, D. Y. et al.,1989, Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase(Lizardi, P. M. et al., 1988, Bio/Technology 6:1197), or any othernucleic acid amplification method, followed by the detection of theamplified molecules using techniques well known to those of skill in theart. These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

In a preferred embodiment of the subject assay, mutations in, or allelicvariants, of an ACE-2 gene from a sample cell are identified byalterations in restriction enzyme cleavage patterns. For example, sampleand control DNA is isolated, amplified (optionally), digested with oneor more restriction endonucleases, and fragment length sizes aredetermined by gel electrophoresis. Moreover, the use of sequencespecific ribozymes (see, for example, U.S. Pat. No. 5,498,531) can beused to score for the presence of specific mutations by development orloss of a ribozyme cleavage site.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the ACE-2 gene anddetect mutations by comparing the sequence of the sample ACE-2 with thecorresponding wild-type (control) sequence. Exemplary sequencingreactions include those based on techniques developed by Maxim andGilbert (Proc. Natl Acad Sci USA (1977) 74:560) or Sanger (Sanger et al(1977) Proc. Nat. Acad. Sci 74:5463). It is also contemplated that anyof a variety of automated sequencing procedures may be utilized whenperforming the subject assays (Biotechniques (1995) 19:448), includingsequencing by mass spectrometry (see, for example PCT publication WO94/16101; Cohen et al. (1996) Adv Chromatogr 36:127-162; and Griffin etal. (1993) Appl Biochem Biotechnol 38:147-159). It will be evident toone skilled in the art that, for certain embodiments, the occurrence ofonly one, two or three of the nucleic acid bases need be determined inthe sequencing reaction. For instance, A-track or the like, e.g., whereonly one nucleic acid is detected, can be carried out.

In a further embodiment, protection from cleavage agents (such as anuclease, hydroxylamine or osmium tetroxide and with piperidine) can beused to detect mismatched bases in RNA/RNA or RNA/DNA or DNA/DNAheteroduplexes (Myers, et al. (1985) Science 230:1242). In general, theart technique of “mismatch cleavage” starts by providing heteroduplexesformed by hybridizing (labelled) RNA or DNA containing the wild-typeACE-2 sequence with potentially mutant RNA or DNA obtained from a tissuesample. The double-stranded duplexes are treated with an agent whichcleaves single-stranded regions of the duplex such as which will existdue to base pair mismatches between the control and sample strands. Forinstance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybridstreated with S1 nuclease to enzymatically digest the mismatched regions.In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treatedwith hydroxylamine or osmium tetroxide and with piperidine in order todigest mismatched regions. After digestion of the mismatched regions,the resulting material is then separated by size on denaturingpolyacrylamide gels to determine the site of mutation. See, for example,Cotton et al (1988) Proc. Natl Acad Sci USA 85:4397; Saleeba et al(1992) Methods Enzymod. 217:286-295. In a preferred embodiment, thecontrol DNA or RNA can be labeled for detection.

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair” enzymes) in defined systems fordetecting and mapping point mutations in ACE-2 cDNAs obtained fromsamples of cells. For example, the mutY enzyme of E. coli cleaves A atG/A mismatches and the thymidine DNA glycosylase from HeLa cells cleavesT at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662).According to an exemplary embodiment, a probe based on an ACE-2sequence, e.g., a wild-type ACE-2 sequence, is hybridized to a cDNA orother DNA product from a test cell(s). The duplex is treated with a DNAmismatch repair enzyme, and the cleavage products, if any, can bedetected from electrophoresis protocols or the like. See, for example,U.S. Pat. No. 5,459,039.

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations or the identity of the allelic variant of apolymorphic region in ACE-2 genes. For example, single strandconformation polymorphism (SSCP) may be used to detect differences inelectrophoretic mobility between mutant and wild type nucleic acids(Orita et al. (1989) Proc Natl. Acad. Sci USA 86:2766, see also Cotton(1993) Mutat Res 285:125-144; and Hayashi (1992) Genet Anal Tech Appl9:73-79). Single-stranded DNA fragments of sample and control ACE-2nucleic acids will be denatured and allowed to renature. The secondarystructure of single-stranded nucleic acids varies according to sequence,the resulting alteration in electrophoretic mobility enables thedetection of even a single base change. The DNA fragments may belabelled or detected with labelled probes. The sensitivity of the assaymay be enhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In a preferredembodiment, the subject method utilizes heteroduplex analysis toseparate double stranded heteroduplex molecules on the basis of changesin electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).

In yet another embodiment the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE) (Myers et al (1985)Nature 313:495). When DGGE is used as the method of analysis, DNA willbe modified to insure that it does not completely denature, for exampleby adding a GC clamp of approximately 40 bp of high-melting GC-rich DNAby PCR. In a further embodiment, a temperature gradient is used in placeof a denaturing agent gradient to identify differences in the mobilityof control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem265:12753).

Examples of other techniques for detecting point mutations or theidentity of the allelic variant of a polymorphic region include, but arenot limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation ornucleotide difference (e.g., in allelic variants) is placed centrallyand then hybridized to target DNA under conditions which permithybridization only if a perfect match is found (Saiki et al. (1986)Nature 324:163); Saiki et al (1989) Proc. Natl Acad. Sci USA 86:6230).Such allele specific oligonucleotide hybridization techniques may beused to test one mutation or polymorphic region per reaction whenoligonucleotides are hybridized to PCR amplified target DNA or a numberof different mutations or polymorphic regions when the oligonucleotidesare attached to the hybridizing membrane and hybridized with labelledtarget DNA.

Alternatively, allele specific amplification technology which depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the mutation or polymorphic region of interest in the centerof the molecule (so that amplification depends on differentialhybridization) (Gibbs et al (1989) Nucleic Acids Res. 17:2437-2448) orat the extreme 3′ end of one primer where, under appropriate conditions,mismatch can prevent, or reduce polymerase extension (Prossner (1993)Tibtech 11:238. In addition it may be desirable to introduce a novelrestriction site in the region of the mutation to create cleavage-baseddetection (Gasparini et al (1992) Mol. Cell Probes 6:1). It isanticipated that in certain embodiments amplification may also beperformed using Taq ligase for amplification (Barany (1991) Proc. Natl.Acad Sci USA 88:189). In such cases, ligation will occur only if thereis a perfect match at the 3′ end of the 5′ sequence making it possibleto detect the presence of a known mutation at a specific site by lookingfor the presence or absence of amplification.

In another embodiment, identification of the allelic variant is carriedout using an oligonucleotide ligation assay (OLA), as described, e.g.,in U.S. Pat. No. 4,998,617 and in Landegren, U. et al., Science241:1077-1080 (1988). The OLA protocol uses two oligonucleotides whichare designed to be capable of hybridizing to abutting sequences of asingle strand of a target. One of the oligonucleotides is linked to aseparation marker, e.g,. biotinylated, and the other is detectablylabeled. If the precise complementary sequence is found in a targetmolecule, the oligonucleotides will hybridize such that their terminiabut, and create a ligation substrate. Ligation then permits the labeledoligonucleotide to be recovered using avidin, or another biotin ligand.Nickerson, D. A. et al. have described a nucleic acid detection assaythat combines attributes of PCR and OLA (Nickerson, D. A. et al., Proc.Natl. Acad. Sci. (U.S.A.) 87:8923-8927 (1990). In this method, PCR isused to achieve the exponential amplification of target DNA, which isthen detected using OLA.

Several techniques based on this OLA method have been developed and canbe used to detect specific allelic variants of a polymorphic region ofan ACE-2 gene. For example, U.S. Pat. No. 5,593,826 discloses an OLAusing an oligonucleotide having 3′-amino group and a 5′-phosphorylatedoligonucleotide to form a conjugate having a phosphoramidate linkage. Inanother variation of OLA described in Tobe et al. ((1996)Nucleic AcidsRes 24: 3728), OLA combined with PCR permits typing of two alleles in asingle microtiter well. By marking each of the allele-specific primerswith a unique hapten, i.e. digoxigenin and fluorescein, each OLAreaction can be detected by using hapten specific antibodies that arelabeled with different enzyme reporters, alkaline phosphatase orhorseradish peroxidase. This system permits the detection of the twoalleles using a high throughput format that leads to the production oftwo different colors.

The invention further provides methods for detecting single nucleotidepolymorphisms in an ACE-2 gene. Because single nucleotide polymorphismsconstitute sites of variation flanked by regions of invariant sequence,their analysis requires no more than the determination of the identityof the single nucleotide present at the site of variation and it isunnecessary to determine a complete gene sequence for each patient.Several methods have been developed to facilitate the analysis of suchsingle nucleotide polymorphisms.

In one embodiment, the single base polymorphism can be detected by usinga specialized exonuclease-resistant nucleotide, as disclosed, e.g., inMundy, C. R. (U.S. Pat. No. 4,656,127). According to the method, aprimer complementary to the allelic sequence immediately 3′ to thepolymorphic site is permitted to hybridize to a target molecule obtainedfrom a particular animal or human. If the polymorphic site on the targetmolecule contains a nucleotide that is complementary to the particularexonuclease-resistant nucleotide derivative present, then thatderivative will be incorporated onto the end of the hybridized primer.Such incorporation renders the primer resistant to exonuclease, andthereby permits its detection. Since the identity of theexonuclease-resistant derivative of the sample is known, a finding thatthe primer has become resistant to exonucleases reveals that thenucleotide present in the polymorphic site of the target molecule wascomplementary to that of the nucleotide derivative used in the reaction.This method has the advantage that it does not require the determinationof large amounts of extraneous sequence data.

In another embodiment of the invention, a solution-based method is usedfor determining the identity of the nucleotide of a polymorphic site.Cohen, D. et al. (French Patent 2,650,840; PCT Appln. No. WO91/02087).As in the Mundy method of U.S. Pat. No. 4,656,127, a primer is employedthat is complementary to allelic sequences immediately 3′ to apolymorphic site. The method determines the identity of the nucleotideof that site using labeled dideoxynucleotide derivatives, which, ifcomplementary to the nucleotide of the polymorphic site will becomeincorporated onto the terminus of the primer.

An alternative method, known as Genetic Bit Analysis or GBA □ isdescribed by Goelet, P. et al. (PCT Appln. No. 92/15712). The method ofGoelet, P. et al. uses mixtures of labeled terminators and a primer thatis complementary to the sequence 3′ to a polymorphic site. The labeledterminator that is incorporated is thus determined by, and complementaryto, the nucleotide present in the polymorphic site of the targetmolecule being evaluated. In contrast to the method of Cohen et al.(French Patent 2,650,840; PCT Appln. No. WO91/02087) the method ofGoelet, P. et al. is preferably a heterogeneous phase assay, in whichthe primer or the target molecule is immobilized to a solid phase.

Recently, several primer-guided nucleotide incorporation procedures forassaying polymorphic sites in DNA have been described (Komher, J. S. etal., Nucl. Acids. Res. 17:7779-7784 (1989); Sokolov, B. P., Nucl. AcidsRes. 18:3671 (1990); Syvanen, A.-C., et al., Genomics 8:684-692 (1990);Kuppuswamy, M. N. et al., Proc. Natl. Acad. Sci. (U.S.A.) 88:1143-1147(1991); Prezant, T. R. et al., Hum. Mutat. 1:159-164 (1992); Ugozzoli,L. et al., GATA 9:107-112 (1992); Nyren, P. et al., Anal. Biochem.208:171-175 (1 993)). These methods differ from GBA TM in that they allrely on the incorporation of labeled deoxynucleotides to discriminatebetween bases at a polymorphic site. In such a format, since the signalis proportional to the number of deoxynucleotides incorporated,polymorphisms that occur in runs of the same nucleotide can result insignals that are proportional to the length of the run (Syvanen, A. -C.,et al., Amer.J. Hum. Genet. 52:46-59 (1993)).

For mutations that produce premature termination of protein translation,the protein truncation test (PTT) offers an efficient diagnosticapproach (Roest, et. al., (1993) Hum. Mol. Genet. 2:1719-21; van derLuijt, et. al., (1994) Genomics 20:14). For PTT, RNA is initiallyisolated from available tissue and reverse-transcribed, and the segmentof interest is amplified by PCR. The products of reverse transcriptionPCR are then used as a template for nested PCR amplification with aprimer that contains an RNA polymerase promoter and a sequence forinitiating eukaryotic translation. After amplification of the region ofinterest, the unique motifs incorporated into the primer permitsequential in vitro transcription and translation of the PCR products.Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis oftranslation products, the appearance of truncated polypeptides signalsthe presence of a mutation that causes premature termination oftranslation. In a variation of this technique, DNA (as opposed to RNA)is used as a PCR template when the target region of interest is derivedfrom a single exon.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one probe nucleic acid,primer set; and/or antibody reagent described herein, which may beconveniently used, e.g., in clinical settings to diagnose patientsexhibiting symptoms or family history of a disease or illness involvingan ACE-2 polypeptide.

Any cell type or tissue may be utilized in the diagnostics describedbelow. In a preferred embodiment a bodily fluid, e.g., blood or urine,is obtained from the subject to determine the presence of a mutation orthe identity of the allelic variant of a polymorphic region of an ACE-2gene. A bodily fluid, e.g, blood, can be obtained by known techniques(e.g. venipuncture). Alternatively, nucleic acid tests can be performedon dry samples (e.g. hair or skin). For prenatal diagnosis, fetalnucleic acid samples can be obtained from maternal blood as described inInternational Patent Application No. WO91/07660 to Bianchi.Alternatively, amniocytes or chorionic villi may be obtained forperforming prenatal testing.

When using RNA or protein to determine the presence of a mutation or ofa specific allelic variant of a polymorphic region of an ACE-2 gene, thecells or tissues that may be utilized must express the ACE-2 gene.Preferred cells for use in these methods include kidney cells andcardiac cells (see Examples). Alternative cells or tissues that can beused, can be identified by determining the expression pattern of thespecific ACE-2 gene in a subject, such as by Northern blot analysis.

Diagnostic procedures may also be performed in situ directly upon tissuesections (fixed and/or frozen) of patient tissue obtained from biopsiesor resections, such that no nucleic acid purification is necessary.Nucleic acid reagents may be used as probes and/or primers for such insitu procedures (see, for example, Nuovo, G. J., 1992, PCR in situhybridization: protocols and applications, Raven Press, NY).

In addition to methods which focus primarily on the detection of onenucleic acid sequence, profiles may also be assessed in such detectionschemes. Fingerprint profiles may be generated, for example, byutilizing a differential display procedure, Northern analysis and/orRT-PCR.

Antibodies directed against wild type or mutatnt ACE-2 polypeptides orallelic variant thereof, which are discussed above, may also be used indisease diagnostics and prognostics. Such diagnostic methods, may beused to detect abnormalities in the level of ACE-2 polypeptideexpression, or abnormalities in the structure and/or tissue, cellular,or subcellular location of an ACE-2 polypeptide. Structural differencesmay include, for example, differences in the size, electronegativity, orantigenicity of the mutant ACE-2 polypeptide relative to the normalACE-2 polypeptide. Protein from the tissue or cell type to be analyzedmay easily be detected or isolated using techniques which are well knownto one of skill in the art, including but not limited to western blotanalysis. For a detailed explanation of methods for carrying out Westernblot analysis, see Sambrook et al, 1989, supra, at Chapter 18. Theprotein detection and isolation methods employed herein may also be suchas those described in Harlow and Lane, for example, (Harlow, E. andLane, D., 1988, “Antibodies: A Laboratory Manual”, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.), which is incorporatedherein by reference in its entirety.

This can be accomplished, for example, by immunofluorescence techniquesemploying a fluorescently labeled antibody (see below) coupled withlight microscopic, flow cytometric, or fluorimetric detection. Theantibodies (or fragments thereof) useful in the present invention may,additionally, be employed histologically, as in immunofluorescence orimmunoelectron microscopy, for in situ detection of ACE-2 polypeptides.In situ detection may be accomplished by removing a histologicalspecimen from a patient, and applying thereto a labeled antibody of thepresent invention. The antibody (or fragment) is preferably applied byoverlaying the labeled antibody (or fragment) onto a biological sample.Through the use of such a procedure, it is possible to determine notonly the presence of the ACE-2 polypeptide, but also its distribution inthe examined tissue. Using the present invention, one of ordinary skillwill readily perceive that any of a wide variety of histological methods(such as staining procedures) can be modified in order to achieve suchin situ detection.

Often a solid phase support or carrier is used as a support capable ofbinding an antigen or an antibody. Well-known supports or carriersinclude glass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, gabbros, andmagnetite. The nature of the carrier can be either soluble to someextent or insoluble for the purposes of the present invention. Thesupport material may have virtually any possible structuralconfiguration so long as the coupled molecule is capable of binding toan antigen or antibody. Thus, the support configuration may bespherical, as in a bead, or cylindrical, as in the inside surface of atest tube, or the external surface of a rod. Alternatively, the surfacemay be flat such as a sheet, test strip, etc. Preferred supports includepolystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

One means for labeling an anti-ACE-2 polypeptide specific antibody isvia linkage to an enzyme and use in an enzyme immunoassay (EIA) (Voller,“The Enzyme Linked Immunosorbent Assay (ELISA)”, Diagnostic Horizons2:1-7, 1978, Microbiological Associates Quarterly Publication,Walkersville, Md.; Voller, et al., J. Clin. Pathol. 31:507-520 (1978);Butler, Meth. Enzymol. 73:482-523 (1981); Maggio, (ed.) EnzymeImmunoassay, CRC Press, Boca Raton, Fl., 1980; Ishikawa, et al., (eds.)Enzyme Immunoassay, Kgaku Shoin, Tokyo, 1981). The enzyme which is boundto the antibody will react with an appropriate substrate, preferably achromogenic substrate, in such a manner as to produce a chemical moietywhich can be detected, for example, by spectrophotometric, fluorimetricor by visual means. Enzymes which can be used to detectably label theantibody include, but are not limited to, malate dehydrogenase,staphylococcal nuclease, delta-5-steroid isomerase, yeast alcoholdehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphateisomerase, horseradish peroxidase, alkaline phosphatase, asparaginase,glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. The detection can be accomplished by colorimetricmethods which employ a chromogenic substrate for the enzyme. Detectionmay also be accomplished by visual comparison of the extent of enzymaticreaction of a substrate in comparison with similarly prepared standards.

Detection may also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the antibodies orantibody fragments, it is possible to detect fingerprint gene wild typeor mutant peptides through the use of a radioimmunoassay (RIA) (see, forexample, Weintraub, B., Principles of Radioimmunoassays, SeventhTraining Course on Radioligand Assay Techniques, The Endocrine Society,March, 1986, which is incorporated by reference herein). The radioactiveisotope can be detected by such means as the use of a gamma counter or ascintillation counter or by autoradiography.

It is also possible to label the antibody with a fluorescent compound.When the fluorescently labeled antibody is exposed to light of theproper wave length, its presence can then be detected due tofluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

The antibody can also be detectably labeled using fluorescence emittingmetals such as ¹⁵²Eu, or others of the lanthanide series. These metalscan be attached to the antibody using such metal chelating groups asdiethylenetriaminetetraacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody ofthe present invention. Bioluminescence is a type of chemiluminescencefound in biological systems in, which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Important bioluminescent compounds for purposes oflabeling are luciferin, luciferase and aequorin.

Moreover, it will be understood that any of the above methods fordetecting alterations in a gene or gene product or polymorphic variantscan be used to monitor the course of treatment or therapy.

In yet other diagnostic methods, the amount of one or more ACE-2 targetpeptide or conversion product thereof is determined. Such amounts can beindicative of the existence of an aberrrant ACE-2 level or activity andcan be predictive of diseases or disorders associated therewith.

8.2. Pharmacogenomics

Knowledge of the particular alteration or alterations, resulting indefective or deficient ACE-2 genes or proteins in an individual (theACE-2 genetic profile), alone or in conjunction with information onother genetic defects contributing to the same disease (the geneticprofile of the particular disease) allows a customization of the therapyfor a particular disease to the individual's genetic profile, the goalof “pharmacogenomics”. For example, subjects having a specific allele ofan ACE-2 gene may or may not exhibit symptoms of a particular disease orbe predisposed of developing symptoms of a particular disease. Further,if those subjects are symptomatic, they may or may not respond to acertain drug, e.g., a specific ACE-2 therapeutic, but may respond toanother. Thus, generation of an ACE-2 genetic profile, (e.g.,categorization of alterations in ACE-2 genes which are associated withthe development of a particular disease), from a population of subjects,who are symptomatic for a disease or condition that is caused by orcontributed to by a defective and/or deficient ACE-2 gene and/or protein(an ACE-2 genetic population profile) and comparison of an individual'sACE-2 profile to the population profile, permits the selection or designof drugs that are expected to be safe and efficacious for a particularpatient or patient population (i.e., a group of patients having the samegenetic alteration).

For example, an ACE-2 population profile can be performed, bydetermining the ACE-2 profile, e.g., the identity of ACE-2 genes, in apatient population having a disease, which is caused by or contributedto by a defective or deficient ACE-2 gene. Optionally, the ACE-2population profile can further include information relating to theresponse of the population to an ACE-2 therapeutic, using any of avariety of methods, including, monitoring: 1) the severity of symptomsassociated with the ACE-2 related disease, 2) ACE-2 gene expressionlevel, 3) ACE-2 mRNA level, and/or 4) ACE-2 protein level. and (iii)dividing or categorizing the population based on the particular geneticalteration or alterations present in its ACE-2 gene or an ACE-2 pathwaygene. The ACE-2 genetic population profile can also, optionally,indicate those particular alterations in which the patient was eitherresponsive or non-responsive to a particular therapeutic. Thisinformation or population profile, is then useful for predicting whichindividuals should respond to particular drugs, based on theirindividual ACE-2 profile.

In a preferred embodiment, the ACE-2 profile is a transcriptional orexpression level profile and step (i) is comprised of determining theexpression level of ACE-2 proteins, alone or in conjunction with theexpression level of other genes, known to contribute to the samedisease, can be measured in many patients at various stages of thedisease.

Pharmacogenomic studies can also be performed using transgenic animals.For example, one can produce transgenic mice, e.g., as described herein,which contain a specific allelic variant of an ACE-2 gene. These micecan be created, e.g, by replacing their wild-type ACE-2 gene with anallele of the human ACE-2 gene. The response of these mice to specificACE-2 therapeutics can then be determined.

8.3. Monitoring of Effects of ACE-2 Therapeutics During Clinical Trials

The ability to target populations expected to show the highest clinicalbenefit, based on the ACE-2 or disease genetic profile, can enable: 1)the repositioning of marketed drugs with disappointing market results;2) the rescue of drug candidates whose clinical development has beendiscontinued as a result of safety or efficacy limitations, which arepatient subgroup-specific; and 3) an accelerated and less costlydevelopment for drug candidates and more optimal drug labeling (e.g.since the use of ACE-2 as a marker is useful for optimizing effectivedose).

The treatment of an individual with an ACE-2 therapeutic can bemonitored by determining ACE-2 characteristics, such as ACE-2 proteinlevel or activity, ACE-2 mRNA level, ACE-2 transcriptional level, and/orlevel of one or more angiotensin or kinetensin conversion products,e.g., Ang.(1-9). This measurements will indicate whether the treatmentis effective or whether it should be adjusted or optimized. Thus, ACE-2can be used as a marker for the efficacy of a drug during clinicaltrials.

In a preferred embodiment, the present invention provides a method formonitoring the effectiveness of treatment of a subject with an agent(e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleicacid, small molecule, or other drug candidate identified by thescreening assays described herein) comprising the steps of (i) obtaininga preadministration sample from a subject prior to administration of theagent; (ii) detecting the level of expression of an ACE-2 protein, mRNA,or genomic DNA and/or level of one or more angiotensin or kinetensinconversion products, e.g., Ang.(1-9) in the preadministration sample;(iii) obtaining one or more post-administration samples from thesubject; (iv) detecting the level of expression or activity of the ACE-2protein, mRNA, or genomic DNA and/or level of one or more angiotensin orkinetensin conversion products, e.g., Ang.(1-9) in thepost-administration samples; (v) comparing the level of expression oractivity of the ACE-2 protein, mRNA, or genomic DNA and/or level of oneor more angiotensin or kinetensin conversion products, e.g., Ang.(1-9)in the preadministration sample with that in the post administrationsample or samples; and (vi) altering the administration of the agent tothe subject accordingly. For example, increased administration of theagent may be desirable to increase the expression or activity of ACE-2to higher levels than detected, i.e., to increase the effectiveness ofthe agent. Alternatively, decreased administration of the agent may bedesirable to decrease expression or activity of ACE-2 to lower levelsthan detected, i.e., to decrease the effectiveness of the agent.

Cells of a subject may also be obtained before and after administrationof an ACE-2 therapeutic to detect the level of expression of genes otherthan ACE-2, to verify that the ACE-2 therapeutic does not increase ordecrease the expression of genes which could be deleterious. This can bedone, e.g., by using the method of transcriptional profiling. Thus, mRNAfrom cells exposed in vivo to an ACE-2 therapeutic and mRNA from thesame type of cells that were not exposed to the ACE-2 therapeutic couldbe reverse transcribed and hybridized to a chip containing DNA fromnumerous genes, to thereby compare the expression of genes in cellstreated and not treated with an ACE-therapeutic. If, for example anACE-2 therapeutic turns on the expression of a proto-oncogene in anindividual, use of this particular ACE-2 therapeutic may be undesirable.

8.4. Use of ACE-2 Molecules as Surrogate Markers

The ACE-2 molecules of the invention are also useful as markers ofdisorders or disease states, as markers for precursors of diseasestates, as markers for predisposition of disease states, as markers ofdrug activity, or as markers of the pharmacogenomic profile of asubject. Using the methods described herein, the presence, absenceand/or quantity of the ACE-2 molecules of the invention may be detected,and may be correlated with one or more biological states in vivo. Forexample, the ACE-2 molecules of the invention may serve as surrogatemarkers for one or more disorders or disease states or for conditionsleading up to disease states. As used herein, a “surrogate marker” is anobjective biochemical marker which correlates with the absence orpresence of a disease or disorder, or with the progression of a diseaseor disorder (e.g., with the presence or absence of a tumor). Thepresence or quantity of such markers is independent of the disease.Therefore, these markers may serve to indicate whether a particularcourse of treatment is effective in lessening a disease state ordisorder. Surrogate markers are of particular use when the presence orextent of a disease state or disorder is difficult to assess throughstandard methodologies (e.g., early stage tumors), or when an assessmentof disease progression is desired before a potentially dangerousclinical endpoint is reached (e.g., an assessment of cardiovasculardisease may be made using cholesterol levels as a surrogate marker, andan analysis of HIV infection may be made using HIV RNA levels as asurrogate marker, well in advance of the undesirable clinical outcomesof myocardial infarction or fully-developed AIDS). Examples of the useof surrogate markers in the art include: Koomen et al. (2000) J. Mass.Spectrom. 35: 258-264; and James (1 994) AIDS Treatment News Archive209.

The ACE-2 molecules of the invention are also useful as pharmacodynamicmarkers. As used herein, a “pharmacodynamic marker” is an objectivebiochemical marker which correlates specifically with drug effects. Thepresence or quantity of a pharmacodynamic marker is not related to thedisease state or disorder for which the drug is being administered;therefore, the presence or quantity of the marker is indicative of thepresence or activity of the drug in a subject. For example, apharmacodynamic marker may be indicative of the concentration of thedrug in a biological tissue, in that the marker is either expressed ortranscribed or not expressed or transcribed in that tissue inrelationship to the level of the drug. In this fashion, the distributionor uptake of the drug may be monitored by the pharmacodynamic marker.Similarly, the presence or quantity of the pharmacodynamic marker may berelated to the presence or quantity of the metabolic product-of a drug,such that the presence or quantity of the marker is indicative of therelative breakdown rate of the drug in vivo. Pharmacodynamic markers areof particular use in increasing the sensitivity of detection of drugeffects, particularly when the drug is administered in low doses. Sinceeven a small amount of a drug may be sufficient to activate multiplerounds of marker (e.g., an ACE-2 marker) transcription or expression,the amplified marker may be in a quantity which is more readilydetectable than the drug itself. Also, the marker may be more easilydetected due to the nature of the marker itself; for example, using themethods described herein, anti-ACE-2 antibodies may be employed in animmune-based detection system for an ACE-2 polypeptide marker, orACE-2-specific radiolabeled probes may be used to detect an ACE-2 mRNAmarker. Furthermore, the use of a pharmacodynanic marker may offermechanism-based prediction of risk due to drug treatment beyond therange of possible direct observations. Examples of the use ofpharmacodynamic markers in the art include: Matsuda et al. U.S. Pat. No.6,033,862; Hattis et al. (1991) Env. Health Perspect. 90: 229-238;Schentag (1999) Am. J. Health-Syst. Pharm. 56 Suppl. 3: S21-S24; andNicolau (1999) Am, J. Health-Syst. Pharm. 56 Suppl. 3: S16-S20.

The ACE-2 molecules of the invention are also useful as pharmacogenomicmarkers. As used herein, a “pharmacogenomic marker” is an objectivebiochemical marker which correlates with a specific clinical drugresponse or susceptibility in a subject (see, e.g., McLeod et al. (1999)Eur. J. Cancer 35(12): 1650-1652). The presence or quantity of thepharmacogenomic marker is related to the predicted response of thesubject to a specific drug or class of drugs prior to administration ofthe drug. By assessing the presence or quantity of one or morepharmacogenomic markers in a subject, a drug therapy which is mostappropriate for the subject, or which is predicted to have a greaterdegree of success, may be selected. For example, based on the presenceor quantity of RNA, or polypeptide (e.g., ACE-2 polypeptide or RNA) forspecific tumor markers in a subject, a drug or course of treatment maybe selected that is optimized for the treatment of the specific tumorlikely to be present in the subject. Similarly, the presence or absenceof a specific sequence mutation in ACE-2 DNA may correlate ACE-2 drugresponse. The use of pharmacogenomic markers therefore permits theapplication of the most appropriate treatment for each subject withouthaving to administer the therapy.

9. Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject having or likely to develop a disorderassociated with aberrant ACE-2 expression or activity or aberrant levelof angiotensin or other target peptide or conversion product thereof,e.g., disorders or diseases associated with an abnormal blood pressure.

9.1. Prophylactic Methods

In one aspect, the invention provides a method for preventing in asubject, a disease or condition associated with an aberrant ACE-2expression or activity or an abnormal amount of target peptide orabnormal blood pressure or abnormal conductance, by administering to thesubject an agent which modulates ACE-2 expression or at least one ACE-2activity. Subjects at risk for such a disease can be identified by adiagnostic or prognostic assay, e.g., as described herein.Administration of a prophylactic agent can occur prior to themanifestation of symptoms characteristic of the ACE-2 aberrancy, suchthat a disease or disorder is prevented or, alternatively, delayed inits progression. Depending on the type of ACE-2 aberrancy, for example,a ACE-2 agonist or ACE-2 antagonist agent can be used for treating thesubject prophylactically. In particular, an ACE-2 therapeutic can beadministered prophylactically in a subject having elevated levels ofangiotensin II, before any other symptoms are present. Such aprophylactic treatment could thus prevent the development of aabnormally high blood pressure. An agonist can also be administeredprophylactically to prevent the development of a kinetensin associatedcondition, e.g., allergies. An antagonist may also be administeredprophylactically to prevent the development of a disorder associatedwith abnormal conductance, such as an arrhythmia. The prophylacticmethods are similar to therapeutic methods of the present invention andare further discussed in the following subsections.

9.2. Therapeutic Methods

In general, the invention provides methods for treating a disease causedby or contributed to by an aberrant ACE-2 activity or an abnormal bloodpressure or abnormal amount of target peptide or angiotensin conversionproduct in a subject, comprising administering to the subject aneffective amount of a pharmaceutical composition comprising a compoundwhich is capable of modulating an ACE-2 activity, such that the diseaseis treated or prevented in the subject. Among the approaches which maybe used to ameliorate disease symptoms involving an aberrant ACE-2activity are, for example, antisense, ribozyme, and triple helixmolecules described above. Examples of suitable compounds include theantagonists, agonists or homologues described in detail herein, as wellas angiotensin conversion products or derivatives thereof.

9.3. Diseases or Conditions that can be Treated or Prevented with ACE-2Therapeutics

As set forth herein, ACE-2 has significant sequence homologies with ACEenzymes and has also been shown to hydrolyze angiotensin I intoAng.(1-9) (see Examples). It has also been described that Ang.(1-9) canfurther be hydrolyzed by ACE into Ang.(1-5). Thus, ACE-2 competes withACE for the substrate angiotensin I and hydrolyzes angiotensin I intoAng.(1-9), instead of angiotensin II (Ang.(1-8)), a potentvasoconstrictor. Angiotensin I can also be converted into otherpeptides, having either vasoconstrictive or anti-hypertensive activity(e.g., Ang.(1-7), see, e.g., Chappel et al. (1998) Hypertension 31:362). Various angiotensin conversion pathways are shown in FIG. 8. Thus,based at least on the fact that ACE-2 has the same substrate as ACE andthat both types of enzymes share significant sequence homologies infunctional domains, ACE-2 therapeutics can be used for similartherapeutic and preventive purposes as ACE, as well as additional ones.ACE-2 therapeutics can thus be used not only for treating or preventingdiseases or disorders associated with an aberrant ACE-2 activity, butalso for treating or preventing diseases or disorders associated with anaberrant ACE activity or activity of any other enzyme that is involvedin the conversion of a substrate of ACE-2, such as angiotensin I. Forexample, ACE-2 therapeutics can be useful for treating diseasesassociated with an abnormal Ang.(1-5), Ang.(1-7) level, orAng.(1-8)(i.e., angiotensin II) level. In a preferred embodiment, anACE-2 antagonist is used for treating or preventing hypertension,hypotension, or arrhythmias and related diseases in a subject.

In certain embodiments, treating or preventing a disease in a subjectincludes administrating to the subject an ACE-2 therapeutic which isalso an ACE therapeutic or administering to the subject both an ACE-2and an ACE therapeutic. For example, the ACE-2 therapeutic is anantagonist of both ACE-2 and ACE. Thus, administration of such acompound to a subject will result in inhibition of hydrolysis ofangiotensin I into angiotensin II as well as into Ang.(1-9).Alternatively, the ACE-2 therapeutic can be an agonist of both ACE-2 orACE.

The identity of the therapeutic compound for use in treating orpreventing individual diseases or disorders can be determined byperforming in vitro and/or in vivo assays. Preferred assays are thoseusing animal models, such as animal models for hypertension. In anillustrative embodiment, a hypertensive rat of the transgenic rat strainTGR(mREN2)27 harboring the murine Ren-2 gene or a transgenic ratcomprising a transgene encoding human angiotensinogen and/or human renin(U.S. Pat. No. 5,731,489) are used. These transgenic rats developfulminant hypertension at an early age (Lee et al. (1996) Am J Physiol270: E919) and are thus useful for testing ACE-2 therapeutics. Suchanimal models can also be used for determining the effect of a specificangiotensin conversion product on a disorder, e.g., hypertension, andthereby allow to predict whether an agonist or an antagonist should beused for treating a specific disease or condition.

Accordingly, in a preferred embodiment, the compounds of the presentinvention are useful for regulating blood pressure and in particulararterial hypertension. The method comprises, e.g., administering to thesubject an effective amount of a pharmaceutical composition comprisingan ACE-2 therapeutic compound. An ACE-2 therapeutic compound can be,e.g., a compound which inhibits the formation of angiotensin II, apotent vasoconstrictor, such as an ACE-2 agonist, which, e.g.,hydrolyzes angiotensin I into Ang.(1-9). Thus, in an illustrativeexample, an ACE-2 antagonist is administered for decreasing bloodpressure and an agonist is administered for increasing blood pressure ina subject.

Blood pressure refers to the pressure exerted by the blood upon thewalls of the blood vessels, e.g., arteries, and is usually measured onthe radial artery by means of a sphygmomanometer, and expressed inmillimeters of mercury. The following ranges of blood pressure areusually used a standard for normal versus abnormal blood pressure: anormal blood pressure corresponds to a diastolic blood pressure of lessthan 85 mm Hg; a high normal blood pressure corresponds to a diastolicblood pressure between 85 and 89 mm Hg; a mild hypertension correspondsto a diastolic blood pressure between 90-104 mm Hg; a moderatehypertension corresponds to a diastolic blood pressure between 105 and114 mm Hg; and severe hypertension corresponds to a diastolic bloodpressure higher than 115 mm Hg. Abnormal blood pressure can also bedetermined based on the systolic blood pressure (when the diastolicpressure is less than 90 mm Hg). Thus, a normal blood pressurecorresponds to a systolic blood pressure of less than 140 mm Hg; aborderline systolic hypertension corresponds to a systolic bloodpressure between 140 and 159 mm Hg; and isolated systolic hypertensioncorresponds to a systolic blood pressure higher than 160 mm Hg. Thisclassification is borrowed from Cecil: Essentials of Medicine, ThirdEdition by Andreoli et al. W. B. Saunders Company (1993).

A diagnosis of hypertension, also referred to herein as “abnormally highblood pressure”, is usually made in an adult over 18 years of age if theaverage of two or more blood pressure measurements on at least twosubsequent visits is 90 mm Hg or higher diastolic or 140 mm Hg systolic.Since children and pregnant women have a lower blood pressure, a bloodpressure over 120/80 (i.e., 120 mm Hg systolic blood pressure/80 mm Hgdiastolic blood pressure), is considered abnormal. Isolated systolichypertension (ISH) refers to a condition in which the systolic bloodpressure is greater than 160 mm Hg and the diastolic blood pressure isless than 85 mm Hg. ISH is associated with enhanced morbidity.

ACE-2 therapeutics can also be used to treat other blood pressurerelated diseases or conditions, e.g., CHF, chronic heart failure, leftventricular hypertrophy, acute heart failure, myocardial infarction, andcardiomyopathy. In a preferred embodiment, ACE-2 therapeutics are usedto treat CHF. CHF is characterized by the inability of the leftventricle to maintain a normal blood pressure. This results in abaroflex-mediated reflex increase in sympathetic discharge, whichstimulates the myocardium to beat faster and stronger, yet increasesperipheral vasoconstriction so that the afterload rises and the load onthe failing myocardium augments (Lionel H. Opie, Drugs for the Heart,Third Edition, W. B. Saunders Co., 1991). Excess adrenergic activityalso results in enhanced activity of the renin-angiotensin system,further increasing peripheral vascular resistance and contributing tofluid retention (edema) by stimulation of the secretion of aldosterone.In addition, angiotensin promotes the release of vasopressin tocontribute to abnormal volume regulation and hyponatremia in severe CHF.Overloading of the left ventricle also results in hypertrophy of theventricular muscle, resulting in a decrease in its contractility,further contributing to the condition. As described in the backgroundsection, vasodilators such as ACE-inhibitors are efficient in treatingCHF and reducing mortality. ACE-inhibitors are particularly preferredtherapeutics for treating CHF since they are able to inhibit thedeleterious neurohumoral viscious circle involvingangiotensin-renin-aldosterone. Thus, it is believed that ACE-2therapeutics, which also modulate angiotensin hydrolysis, will also beuseful for treating and preventing CHF.

Since ACE-2 has been found to hydrolyze neurotensin, ACE-2 therapeuticscan be used for treating or preventing conditions associated withneurotensin. Neurotensin, is a 13 amino acid peptide having thefollowing amino acid sequence:pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu (SEQ ID NO:107) isa putative neurotransmitter/neuromodulator in the central and peripheralnervous system. It has been shown to decrease blood pressure in ratsafter i.v. injection (Di Paola and Richelson (1990) Eur. J. Pharmacol.175: 279), to increase coronary blood flow in open-chest dogs (Bauer etal. (1995) J. Cardiovasc. Pharmacol. 25:756), and to inhibit endogenousnorepinephrine releas in rats during periarterial nerve stimulation(Tsuda et al. (1993) Am. J. Hypertens. 6:473). In a rat failure model(moncrotaline treated rats), neurotensin levels are decreased in bothventricles. Thus, based on these results, modulation of neurotensin andconversion products thereof can be used for modulating blood pressure.

In another embodiment, ACE-2 therapeutics may be used to treat adisorder associated with abnormal conductance, such as an arrhythmia.

In another embodiment, ACE-2 therapeutics may be used to treat adisorder such as azotemia, renal disease, renal failure, glomerulardisease, glomerulonephritis (vasculitis), nephritis, acute tubularnecrosis, proteinuria, hematuria, pyuria, pyelonephritis, polyuria,fluid and electrolyte (e.g., sodiuma nd patassium) disturbances,hypovolemia, hyponatremia, hypematremia, hypokalemia (Liddle's Syndrome,Bartter's Syndrome), hyperkalemia (Gordon's Syndrome), acidosis,alkalosis and hyperchloremic metabolic disorders.

Since neurotensin is found mainly in gut endocrine cells of the ileumand is released following a meal, ACE-2 therapeutics could be used fordigestive purposes, and for treating and/or preventing disordersrelating to digestion.

In another embodiment, the invention provides methods for regulatingcell proliferation, such as smooth cell proliferation. Smooth musclecell proliferation in the intima of muscular arteries is a primary causeof vascular stenosis in atherosclerosis, after vascular surgery, andafter coronary angioplasty. Several animal studies have indicated thatthe renin-angiotensin system plays an important role in this vascularresponse to injury. In particular, it has been shown that chronictreatment with ACE inhibitors reduced myointimal thickening followingballoon injury in rat carotid artery or aorta (Powell et al. (1991) J.Am. Coll. Cardiol. 17:137B42B). The stimulatory effect of angiotensin IIon cell growth and replication in the cardiovascular system, which mayresult in myocardial hypertrophy and hypertrophy or hyperplasia ofconduit and resistance vessels in certain subjects is mediated throughangiotensin II receptors (subtype AT1) (Rosendorff C. (1996) J. Am.Coll. Cardiol.28: 803). The importance of ACE in atherosclerosis isfurther described, e.g, in Malik et al. (1997) Am. Heart J. 134:514. Ithas also been shown, that angiotensin caused myocyte hypertrophy andfibroblast proliferation associated with the induction of mRNA forseveral early response genes (c-fos, c-jun, jun B, Egr-1 and c-myc),angiotensinogen and transforming growth factor beta (TGFβ) (Rosendorff(1996) J. Am. Coll. Cardiol. 28: 803-12; Paquet et al. (1990) J.Hypertens.: 8: 565-72). Accordingly, in one embodiment, the inventionprovides a method for reducing or inhibiting smooth muscle cellproliferation, comprising administering to a subject an efficient amountof a composition comprising an ACE-2 therapeutic. In one embodiment, thetherapeutic is administered systemically. However, the ACE-2 therapeuticcan also be administered locally, e.g., at a site of vascular injury.

ACE-2 therapeutics can further be used in treating kidney diseases ordisorders. Angiotensin and ACEs are important in the development and forthe maintenance of the functional and structural integrity of the adultkidney (see, e.g, Hilgers et al. (1997) Semin. Nephrol. 17:492). Chronicrenal disease evolves to end-stage renal failure through events,including enhanced intraglomerular pressure and plasma proteinultrafiltration, mediated at least in part by angiotensin II. It hasbeen reported that ACE inhibitors reduce intracapillary pressure andameliorate glomerular size-selective function (see, e.g., Ruggenenti andRemuzzi (1997) Curr. Opin. Nephrol. Hypertens. 6:489). Thus, based atleast in part on the fact that ACE-2 is expressed in kidney and ishomologous to ACE, ACE-2 therapeutics an be used for treating andpreventing renal diseases.

The role of angiotensin inhibitors in the release of norepinephrine fromthe terminal adrenergic neurons leads to the proposal that angiotensininhibitors should be useful for treating various other hyperadrenergicstates, such as acute myocardial infarction (AMI) and some ventriculararrthythmias.

The invention further provides methods for treating kinetensinassociated conditions. As described herein, ACE-2 cleaves the C-terminalamino acid (leucine) from kinetensin. Kinetensin is a nine amino acidpeptide having SEQ ID NO:23 which has been reported to induce adose-dependent release of histamine from mast cells, as well as induce adose-dependent increase in vascular permeability when injectedintradermally (Sydborn et al. (1989) Agents Actions 27: 68) into rats.Accordingly, modulating the plasma and/or tissue level of kinetensin,such as by modulating the hydrolysis of the C-terminal amino acid fromkinetensin, should be useful for treating conditions that are caused by,or contributed to by, an abnormal kinetensin level. Such conditionsinclude those caused by, or contributed to by, an abnormal histaminerelease from mast cells and/or by an abnormal vascular permeability.Since excessive histamine release is associated with local or systemicallergic reactions, including exzema, asthma, anaphylactic shock, ACE-2therapeutics are believed to be useful for treating these conditions.

In another preferred embodiment, the invention provides a method fordecreasing or inhibiting an inflammatory reaction. In fact, based atleast in part on the homology between ACE and ACE-2 and the fact thatACE is capable of hydrolysing polypeptides other than angiotensin I,such as kinins, e.g., bradykinin, ACE-2 is likely to hydrolyze kinins.In fact, ACE-2 hydrolyzes bradykinin (see Examples). Kinins (e.g.,bradykinin and kallidin) are generally involved in inflammation. Infact, part of the initiation process of an inflammatory reaction ismediated by peptide kinins, such as bradykinin, which are liberated bykallikrein proteases upon tissue destruction. The kinins or otherpeptide messengers, act on specific cell receptors at the inflammationsite to activate the phospholipase enzymes A2 and/or C, to initiate thearachidonate cascade.

Bradykinins are involved in inflammatory reactions on various tissues.For example, it has been found that bradykinin is produced ininflammatory reactions in the intestine, provoking contraction of smoothmuscle and secretion of fluid and ions. The existence of specificbradykinin receptors in the mucosal lining of the intestine and inintestinal smooth muscle is demonstrated by Manning et al. (Nature 229:256 (1982)), showing the influence of bradykinin in very lowconcentrations upon fluid and ion secretion. Thus, the invention can beused to treat inflammatory reactions in the intestine.

Similarly, the compounds of the present invention are also expected tobe effective in treating other diseases or conditions such as SIRS(Systemic Inflammatory Response Syndromes)/sepsis, polytrauma,inflanmmatory bowel disease, acute and chronic pain, bone destruction inrheumatoid and osteo arthritis and periodontal disease, dysmenorrhea,premature labor, brain edema following focal injury, diffuse axonalinjury, stroke, reperfusion injury and cerebral vasospasm aftersubarachnoid hemorrhage, allergic disorders including asthma, adultrespiratory distress syndrome, wound healing and scar formation.

Furthermore, the invention also provides analgesic methods. In fact,bradykinin is known to be one of the most potent naturally occurringstimulators of C-fiber afferents mediating pain. The production ofbradykinin results in pain at the site of the pathological condition,and the overproduction intensifies the pain directly or via stimulationby bradykinin of the activation of the arachidonic acid pathway whichproduces prostaglandins and leukotrienes, more distal mediators ofinflammation (Handbook of Experimental Pharmacology, Vol.25,Springer-Verlag (1969), and Vol. 25 Supplement (1979); Stewart, in“Mediators of the Inflammatory Process,” Henson and Murphy, eds.,Elsevier,(1989)). For example, direct application of bradykinin todenuded skin or intra-arterial or visceral injection results in thesensation of pain in animals and in man. Kinin-like materials have beenisolated from inflammatory sites produced by a variety of stimuli. Inaddition, bradykinin receptors have been localized to nociceptiveperipheral nerve pathways and bradykinin has been demonstrated tostimulate central fibers mediating pain sensation. Bradykinin has alsobeen shown to be capable of causing hyperalgesia in animal models ofpain. (Burch et al., J. Med. Chem., 30:237-269 (1990) and Clark, W. G.Handbook of Experimental Pharmacology, Vol. XXV: Bradykinin, kallidin,and kallikrein. Erdo, E. G. (ed.), 311-322 (1979)). Furthermore, anumber of studies have demonstrated that bradykinin antagonists arecapable of blocking or ameliorating both pain as well as hyperalgesia inboth animals and man (Ammons, W. S. et al. The American PhysiologicalSociety, 0363-6119 (1985); Clark, Handbook of Experimental Pharmacology,Vol. XXV: Bradykinin, kallidin, and kallikrein. Erdo, E. G. (ed.),311-322 (1979); Costello al., European Journal of Pharmacology,171:259-263 (1989); Laneuville et al. European Journal of Pharmacology,137:281-285 (1987); Steranka et al, European Journal of Pharmacology,16:261-262 (1987); and Steranka et al, Neurobiology, 85:3245-3249(1987)). Similarly, Whalley et al, in Naunyn Schmiederberg's Arch.Pharmacol.,336:652-655 (1987) have demonstrated that bradykininantagonists are capable of blocking bradykinin-induced pain in a humanblister base model. Thus, the compositions of the invention comprisingan ACE-2 agonist therapeutics can be applied topically to hydrolyze andthereby inactive bradykinin and/or related kinins to thereby inhibit orreduce pain in burned skin, e.g. in severely burned patients in whomlarge doses of narcotics are required over long periods of time and forthe local treatment of relatively minor burns or other forms of localskin injury.

Similarly, the production of bradykinin seems to be associated with thepain in angina and myocardial ischemia (Kimura et al., Amer. Heart J.85: 635 (1973); Staszewska-Barczak et al., Cardiovasc. Res. 10: 314(1976)). Thus, ACE-2 therapeutics could be used to relieve pain insubjects suffering from angina or myocardial ischemia by degradation ofbradykinin.

The use of the compounds of the invention for reducing or inhibitingpain have significant advantages over currently accepted therapeuticapproaches to analgesia. In fact, while mild to moderate pain can bealleviated with the use of nonsteroidal anti-inflammatory drugs andother mild analgesics, severe pain such as that accompanying surgicalprocedures, burns and severe trauma requires the use of narcoticanalgesics. These drugs carry the limitations of abuse, potential,physical and psychological dependence, altered mental status andrespiratory depression which significantly limit their usefulness. Onthe contrary, the compounds of the invention, i.e., ACE-2 agonisttherapeutics, are likely to be devoid of such undesirable secondaryeffects.

Other disease states in which ACE-2 agonist therapeutics can be usefulinclude in the treatment of burns, perioperative pain, migraine andother forms of pain, shock, central nervous system injury, rhinitis,premature labor, etc. Yet other diseases or conditions in whichbradykinin is overproduced and in which ACE-2 agonist therapeuticscapable of inactivating bradykinin can be useful include pathologicalconditions such as septic (Robinson et al., Am. J. Med. 59: 61 (1975))and hemorrhagic (Hirsch et al., J. Surg. Res. 17: 147 (1974)) shock,anaphylaxis (Collier and James, J. Physiol. 160: 15P (1966)), arthritis(Jasani et al., Ann. Rheum. Dis. 28: 497 (1969); Hamberg et al., AgentsActions 8: 50( 1978); Sharma et al., Arch. Int. Pharmacodyn. 262: 279(1983)), rhinitis (Proud et al., J. Clin. Invest. 72: 1678 (1983);Naclerio et al., Clin. Res. 33: 613A (1985)), asthma (Christiansen etal., J. Clin. Invest. 79: 188 (1987)), inflammatory bowel disease(Zeitlin and Smith, Gut 14: 133 (1973)), sarcoidosis (Cecil: Essentialsof Medicine, Third Edition by Andreoli et al. W. B. Saunders Company(1993)), and certain other conditions including acute pancreatitis,post-gastrectomy dumping syndrome, carcinoid syndrome, migraine, andhereditary angioedema.

Furthermore, bradykinin and bradykinin-related kinins are not onlyproduced endogenously, but may also be injected into an animal, e.g., ahuman, via stings or bites. It is known that insects such as hornets andwasps inject bradykinin related peptides that cause pain, swelling andinflammation. Accordingly, the method provides methods and compounds fortreating insect stings or bites, comprising administering either locallyor systemically to a subject having an insect bite or sting an ACE-2agonist therapeutic, to thereby relieve the pain and reduce theinflammation.

Based at least on the presence of ACE-2 in testis, ACE-therapeuticscould also have a utility in treating infertility or other disordersrelating to gamete maturation.

In addition, it has been discovered that ACE inhibitors are useful intreating cognitive disorders [German Application No. 3901-291-A,published Aug. 3, 1989]. Accordingly, ACE-2 antagonists of the inventionare also likely to be useful in treating cognitive disorders.

9.4. Effective Dose

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining The Ld₅₀ (The Dose Lethal To 50% Of ThePopulation) And The Ed₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD_(50/)ED₅₀.Compounds which exhibit large therapeutic induces are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

9.5. Formulation and Use

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers or excipients. Thus, the compoundsand their physiologically acceptable salts and solvates may beformulated for administration by, for example, injection, inhalation orinsufflation (either through the mouth or the nose) or oral, buccal,parenteral or rectal administration.

For such therapy, the compounds of the invention can be formulated for avariety of loads of administration, including systemic and topical orlocalized administration. Techniques and formulations generally may befound in Remmington's Pharmaceutical Sciences, Meade Publishing Co.,Easton, Pa. For systemic administration, injection is preferred,including intramuscular, intravenous, intraperitoneal, and subcutaneous.For injection, the compounds of the invention can be formulated inliquid solutions, preferably-in physiologically compatible buffers suchas Hank's solution or Ringer's solution. In addition, the compounds maybe formulated in solid form and redissolved or suspended immediatelyprior to use. Lyophilized forms are also included.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., ationd oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to givecontrolled release of the active compound. For buccal administration thecompositions may take the form of tablets or lozenges formulated inconventional manner. For administration by inhalation, the compounds foruse according to the present invention are conveniently delivered in theform of an aerosol spray presentation from pressurized packs or anebuliser, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration bile salts and fusidic acidderivatives. in addition, detergents may be used to facilitatepermeation. Transmucosal administration may be through nasal sprays orusing suppositories. For topical administration, the oligomers of theinvention are formulated into ointments, salves, gels, or creams asgenerally known in the art. A wash solution can be used locally to treatan injury or inflammation to accelerate healing.

In clinical settings, a gene delivery system for the therapeutic ACE-2gene can be introduced into a patient by any of a number of methods,each of which is familiar in the art. For instance, a pharmaceuticalpreparation of the gene delivery system can be introduced systemically,e.g., by intravenous injection, and specific transduction of the proteinin the target cells occurs predominantly from specificity oftransfection provided by the gene delivery vehicle, cell-type ortissue-type expression due to the transcriptional regulatory sequencescontrolling expression of the receptor gene, or a combination thereof.In other embodiments, initial delivery of the recombinant gene is morelimited with introduction into the animal being quite localized. Forexample, the gene delivery vehicle can be introduced by catheter (seeU.S. Pat. No. 5,328,470) or by stereotactic injection (e.g., Chen et al.(1994) PNAS 91: 3054-3057). An ACE-2 gene, such as any one of thesequences represented in the group consisting of SEQ ID NOS 1 and 3 or asequence homologous thereto can be delivered in a gene therapy constructby electroporation using techniques described, for example, by Dev etal. ((1994) Cancer Treat Rev 20:105-115).

The pharmaceutical preparation of the gene therapy construct or compoundof the inventioncan consist essentially of the gene delivery system inan acceptable diluent, or can comprise a slow release matrix in whichthe gene delivery vehicle or compound is imbedded. Alternatively, wherethe complete gene delivery system can be produced intact fromrecombinant cells, e.g., retroviral vectors, the pharmaceuticalpreparation can comprise one or more cells which produce the genedelivery system.

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

10. Kits

The invention further provides kits for use in diagnostics or prognosticmethods or for treating a disease or condition associated with anaberrant ACE-2 activity, ACE-2 target peptide level, or angiotensin orkinetensin conversion product level. The invention also provides kitsfor determining which ACE-2 therapeutic should be administered to asubject. The invention encompasses kits for detecting the presence ofACE-2 mRNA or protein in a biological sample or for determining thepresence of mutations or the identity of polymorphic regions in an ACE-2gene. For example, the kit can comprise a labeled compound or agentcapable of detecting ACE-2 protein or mRNA in a biological sample; meansfor determining the amount of ACE-2 in the sample; and means forcomparing the amount of ACE-2 in the sample with a standard. Thecompound or agent can be packaged in a suitable container. The kit canfurther comprise instructions for using the kit to detect ACE-2 mRNA orprotein.

In one embodiment, the kit comprises a pharmaceutical compositioncontaining an effective amount of an ACE-2 therapeutic and instructionfor use in treating or preventing hypertension. In another embodiment,the kit comprises a pharmaceutical composition comprising an effectiveamount of an ACE-2 agonist therapeutic and instructions for use intreating insect bites. Generally, the kit comprises a pharmaceuticalcomposition comprising an effective amount of an ACE-2 agonist orantagonist therapeutic and instructions for use as an analgesic.

Yet other kits can be used to determine whether a subject has or islikely to develop a disease or condition associated with an aberrantACE-2 activity. Such a kit can comprise, e.g., one or more nucleic acidprobes capable of hybridizing specifically to at least a portion of anACE-2 gene or allelic variant thereof, or mutated form thereof.

11. Additional Uses for ACE-2 Proteins and Nucleic Acids

The ACE-2 nucleic acids of the invention can further be used in thefollowing assays. In one embodiment, the human ACE-2 nucleic acid havingSEQ ID NO:1 or a portion thereof, or a nucleic acid which hybridizesthereto can be used as a chromosomal marker in genomic linkage analysis.Human ACE-2 has been localized to chromosome Xp21-22. Comparison of thechromosomal location of the ACE-2 gene with the location of chromosomalregions which have been shown to be associated with specific diseases orconditions, e.g., by linkage analysis (coinheritance of physicallyadjacent genes), can be indicative of diseases or conditions in whichACE-2 may play a role. A list of chromosomal regions which have beenlinked to specific diseases can be found, for example, in V. McKusick,Mendelian Inheritance in Man (available on line through Johns HopkinsUniversity Welch Medical Library) and athttp://www3.ncbi.nlm.nih.gov/Omim/(Online Mendelian Inheritance in Man).Furthermore, the ACE-2 gene can also be used as a chromosomal marker ingenetic linkage studies involving genes other than ACE-2.

If the ACE-2 gene is shown to be localized in a chromosomal region whichcosegregates, i.e., which is associated, with a specific disease, thedifferences in the cDNA or genomic sequence between affected andunaffected individuals are determined. The presence of a mutation insome or all of the affected individuals but not in any normalindividuals, will be indicative that the mutation is likely to becausing or contributing to the disease.

The present invention is further illustrated by the following exampleswhich should not be construed as limiting in any way. The contents ofall cited references (including literature references, issued patents,published patent applications as cited throughout this application arehereby expressly incorporated by reference. The practice of the presentinvention will employ, unless otherwise indicated, conventionaltechniques of cell biology, cell culture, molecular biology, transgenicbiology, microbiology, recombinant DNA, and immunology, which are withinthe skill of the art Such techniques are explained fully in theliterature. See, for example, Molecular Cloning A Laboratory Manual,2^(nd) Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring HarborLaboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glovered., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis etal. U.S. Pat. No: 4,683,195; Nucleic Acid Hybridization(B. D. Hames & S.J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S.J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R.Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B.Perbal, A Practical Guide To Molecular Cloning (1984); the treatise,Methods In Enzymology (Academic Press, Inc., N.Y.); Gene TransferVectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987,Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155(Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology(Mayer and Walker, eds., Academic Press, London, 1987); Handbook OfExperimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell,eds., 1986); Manipulating the Mouse Embryo, (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1986).

EXAMPLES

1. Cloning and Analysis of Human ACE-2

A full length cDNA encoding human ACE-2 was isolated as follows. A cDNAlibrary was prepared from a human heart of a subject who had congestiveheart failure. Random sequencing of clones from the library identified a1.6 kb clone that has homology to human angiotensin converting enzyme,and which is thus referred to as ACE-2.

5′ RACE was used to clone the 5′ end of the ACE-2 gene. RACE wasperformed using Clontech's Marathon cDNA Amplification Kit. First strandcDNA synthesis was performed using the cDNA synthesis primer suppliedwith the kit and 1 μg poly A⁺ RNA prepared from the heart of a 43 yearold woman with an idiopathic cardiomyopathy using 100 u MMLV reversetranscriptase. Second strand cDNA synthesis was then performed using thesecond strand enzyme cocktail of the Clontech kit. The Marathon cDNAadaptor was ligated to the double stranded cDNA with T4 DNA ligase. Agene specific primer was designed starting about 400 bp downstream ofthe 5′ end of the 1.6 kb ACE-2 clone. The primer had the nucleotidesequence 5′ CAC AGG TTC CAC CAC CCC AAC TAT CTC 3′ (SEQ ID NO:13, whichcorresponds to nucleotides 1528-1554 of SEQ ID NO:1) and thehybridization temperature (Tm) used was 62.7° C. (GC content of 55%).The gene specific primer and an adaptor primer were used for 5′ RACEusing the Advantage Klentaq polymerase mix under the followingconditions: 1 cycle at 94 ° C. for 2 minutes; 35 cycles of 94° C. for 30sec., 60° C. for 45 sec., 72° C. for 30 sec.; and 1 cycle at 72° C. for5 minutes. A comparison of the partial ACE-2 clone with the previouslycloned ACE genes indicated that about 1000 bp were necessary to have thefull length clone if both genes have a similar length. RACE products ofthe expected size were obtained. These products were Southern blottedand shown to hybridize with a probe, corresponding to nucleotides 1152to 1318 of SEQ ID NO:1, from the partial ACE-2 clone. The RACE productswere run on a 1.2% agarose gel, the expected size fragments werevisualized, excised and purified using the Jetsorb Gel Extraction kit(Genomed). The fragments were then ligated into the TA cloning vectorpCR2.1 (InVitrogen). A clone containing a 1.6 kb insert was sequencedand found to have the 5′ end of the gene.

A full length ACE encoding DNA was then prepared as follows. AnEcoRI-BamHI fragment of the 5′ RACE clone and a BamHI-NotI fragment ofthe partial clone containing the 3′ end of the ACE-2 cDNA describedabove were ligated into the EcoRI-NotI sites of pCDNA3.1+vector(InVitrogen). The clones were analyzed by restriction mapping andsequencing. This confirmed that full length cDNA clones encoding humanACE were obtained.

The cDNA described herein encoding ACE-2 is 3396 nucleotides long andhas the nucleotide sequence shown in FIG. 1 and set forth in SEQ IDNO:1. A nucleic acid comprising this cDNA has been deposited at theAmerican Type Culture Collection (12301 Parklawn Drive, Rockville, Md.)on Dec. 3, 1997 has been assigned ATCC Designation No. 209510. This cDNAhas an open reading frame from nucleotide 82 to nucleotide 2496 of SEQID NO: I which is set forth in SEQ ID NO:3 and encodes a protein of 805amino acids having the amino acid sequence shown in FIG. 1 and set forthin SEQ ID NO:2. The ACE-2 protein having SEQ ID NO:2 contains ahydrophobic signal sequence from amino acid 1 to amino acid 18. Thus,the mature ACE-2 protein has the amino acid sequence from about aminoacid 19 to amino acid 805 of SEQ ID NO:2. The presence of the signalpeptide indicates that the ACE-2 protein is secreted and/or membranebound.

The ACE-2 protein further comprises a Zinc binding domain, from aboutamino acid 374 to amino acid 378 of SEQ ID NO:2 which is encoded by thenucleotide sequence from nucleotide 1201 to nucleotide 1215 of SEQ IDNO:1. The ACE-2 protein also comprises a hydrophobic transmembranedomain corresponding to amino acids 741 to amino acid 765 of SEQ ID NO:2which is encoded by the nucleotide sequence from nucleotide 2302 tonucleotide 2376 of SEQ ID NO:1. This transmembrane domain could bemediating membrane attachment of the ACE-2 to a cell membrane.

A BLAST search (Altschul et al. (1990) J. Mol. Biol. 215:403) of thenucleic acid and the amino acid sequences of ACE-2 revealed that ACE-2has significant homology to portions of angiotensin converting enzymes.An alignment of the amino acid sequence of human ACE-2 having SEQ IDNO:2 with human testicular ACE (SEQ ID NO:4; GenBank Accession No.P22966), murine testicular ACE (SEQ ID NO:5; GenBank Accession No.P22967), rabbit testicular ACE (SEQ ID NO:6; GenBank Accession No.P22968), human endothelial ACE (SEQ ID NO:7; GenBank Accession No.P12821; U.S. Pat. No. 5,539,045 by Soubrier et al.; and described inSoubrier et al. (1988) Proc. Natl. Acad. Sci. USA 85:9386), murineendothelial ACE (SEQ ID NO:8; GenBank Accession No. P09470), ratendothelial ACE (SEQ ID NO:9; GenBank Accession No. P47820) and rabbitendothelial ACE (SEQ ID NO:10; GenBank Accession No. P12822) is shown inFIG. 2. The alignment was performed using CLUSTAL W (1.7). Thisalignment shows that the zinc binding domain is conserved among all ACEproteins. Other regions of the ACE-2 disclosed herein, such as theN-terminal region, are significantly different from the other ACEproteins. As further described herein, the previously described ACEproteins exist in two forms, a long form, referred to as endothelialACE, and a short form, referred to as testicular ACE. Endothelial ACE isexpressed in various tissues, whereas testicular ACE is expressedpredominantly in developing sperm cells in the testis. Both of theseforms derive from the same gene by alternative transcription initiation.The testicular ACE is in fact encoded by a mRNA whose transcriptioninitiation site is located in an intron of the ACE gene. Thus, exceptfor the first 67 N-terminal residues (including the 31 amino acid longsignal peptide), human testis ACE is identical to the second half of thehuman endothelial ACE (Ehlers et al. (1989) Proc. Natl. Acad. Sci. USA86:7741). Furthermore, the previously cloned ACE proteins from differentspecies have a significant homology to each other, which is strongerthan the homology of ACE-2 to any of the ACE proteins. Thus, the ACE-2protein is encoded by a newly identified gene, having sequencesimilarities with some regions of the genes encoding the previouslydescribed ACE proteins, such as in the zinc binding domain.

The amino acid sequence comparison indicates that ACE-2 having SEQ IDNO:2 has the highest overall similarity to the human testicular ACE andthat it is 42.9% identical and 62% similar to the amino acid sequence ofhuman testicular ACE. The cDNAs encoding human testicular ACE and ACE-2(SEQ ID NO:1) have an overall identity of 50.8%.

FIG. 3 shows an amino acid alignment of the ACE-2 protein having SEQ IDNO:2 and ACE proteins from Drosophila Melanogaster (SEQ ID NO:11;GenBank Accession No. Q10714) and C. Elegans (SEQ ID NO:12; GenBankAccession No. U56966), as well as human testicular and endothelial ACE.This alignment indicates that ACE-2 has a certain degree of homologywith the Drosophila ACE protein, in particular in the zinc bindingdomain. However, ACE-2 does not have any significant homology with theC. elegans ACE protein.

Thus, based on the results of the BLAST analysis, ACE-2 is likely to bea second member of a novel family of angiotensin converting enzymes.

The BLAST analysis of GenBank with ACE-2 nucleic acid also indicatedhomologies of portions of human ACE-2 with the following ESTs: TABLE IIHomologies of hu ACE-2 cDNA Sequence with EST Sequences Nucleotides ofAccession No. Species SEQ ID NO: 1 % Identity AA397955 human 2759-3202(3′ UTR) 99% AA420969 human 2936-3368 (3′ UTR) 99% AA162058 mouse 457-1012 (coding region) 87% AA416585 human 2985-3368 (3′ UTR) 100% AA421125 human 2987-3287 (3′ UTR) 100%  AA072298 mouse 1485-1742 (codingregion) 83%

Among these ESTs, only AA162058 was annotated in GenBank as beinghomologous to ACE. A 208 bp fragment of a gene having Accession No.Q04027, annotated as human angiotensin converting enzyme, is 61%identical to nucleotides 1144-1353 of human ACE-2 cDNA having SEQ ID NO:1.

2. Tissue Distribution of ACE-2

A 167 bp fragment of human ACE-2 cDNA, corresponding to nucleotides1152-1318 of SEQ ID NO:1, was labeled with ³²P using the MultiprimeLabeling System from Amersham and hybridized at 10⁶ cpm/ml to HumanMultiple Tissue Northern blots from Clontech overnight at 65° C. inNylon Wash. The blots were then washed three times for 30 minutes at 65°C. in 0.5× Nylon Wash. The results indicated that the ACE-2 probehybridized to a mRNA of about 4 kb in kidney, heart, and testis. Theresults indicate that ACE-2 has a more specific tissue distribution thandoes endothelial ACE which is produced by many somatic tissues.

mRNA level of ACE-2 was also determined in a normal heart and comparedto that of a heart of a congestive heart failure patient. For this, a167 bp probe, corresponding to nucleotides 1152-1318 of SEQ ID NO:1,from the ACE homolog was hybridized to heart RNA from a normal and from3 congestive heart failure patients, by Northern blot hybridization. 10μg RNA was run per lane on a 1.2 % MOPS/fornaldehyde gel, which was thentransferred to Hybond N (Amersham) in 10×SSC and crosslinked in aStratalinker (Stratagene). The blot was hybridized overnight at 65° C.with 1×10⁶ cpm/ml of Nylon wash. The ACE-2 probe hybridizes to thenormal heart sample and to two of the three congestive heart failuresamples

3. In Situ Analysis of ACE-2 mRNA and Protein

In situ hybridization of a human ACE-2 probe to human and monkey tissuesdemonstrated the presence of ACE-2 mRNA in endothelial cells and focallyin normal and hypertrophic myocytes in human heart. In monkey kidneys,ACE-2 mRNA was detected in proximal convoluted tubules.

For studying ACE-2 protein, five rabbit polyclonal anti-peptideantibodies were generated against human ACE-2: I82283M, which isdirected against amino acids 51-69 of SEQ ID NO:2, i.e., NTN ITE ENV QNMNNA GDK W (SEQ ID NO:25); 182284M, which is directed against amino acids194-214 of SEQ ID NO:2, i.e., NHY EDY GDY WRG DYE VNG VDG (SEQ IDNO:26); K70417K, which is directed against amino acids 489-508 of SEQ IDNO:2, i.e, EPV PHD ETY CDP ASL FHV SN (SEQ ID NO:27); K70418M, which isdirected against amino acids 704-723 of SEQ ID NO:2, i.e., IRM SRS RINDAF RLN DNS LE (SEQ ID NO:28); and K70419M, which is directed againstamino acids 785-802 of SEQ ID NO:2, i.e, DIS KGE NNP GFQ NTD DVQ (SEQ IDNO:29). All five antibodies are functional for Western blotting andantibody I82283M is particularly efficient in immunhistochemistry.

ACE-2 protein was evaluated in rat, human and monkey tissues using anantibody generated against a fragment of the human peptide. In rat andhuman heart, ACE-2 protein was apparent in endothelial cells. In normalhuman kidneys, ACE-2 was limited to endothelial cells (arterial/venous).In addition to endothelial cell expression, ACE-2 was detected invascular smooth muscle cells of abnormal renal vessels and damaged(sclerotic) glomeruli of hypertensive human kidneys. In clinicallyhealthy monkey kidneys, ACE-2 protein was found in endothelial cells,epithelial cells of Bowman's capsule and proximal tubules.

4. Expression of Recombinant ACE-2 in COS Cells

This example describes a method for producing recombinant full lengthhuman ACE-2 in a mammalian expression system.

An expression construct containing a nucleic acid encoding a full lengthhuman ACE-2 protein, or a soluble ACE-2 protein which is devoid of thesignal sequence and the transmembrane domain was constructed as follows.A nucleic acid encoding the full length human ACE-2 protein or thesoluble ACE-2 protein is obtained by reverse transcription (RT-PCR) ofmRNA extracted from human cells expressing ACE-2, e.g., human kidneycells using PCR primers based on the sequence set forth in SEQ ID NO:1.The PCR primers further contain appropriate restriction sites forintroduction into the expression plasmid. The amplified nucleic acid isthen inserted in a eukaryotic expression plasmid such as pcDNAI/Amp(InVitrogen) containing: 1) SV40 origin of replication, 2) ampicillinresistance gens, 3) E. coli replication origin, 4) CMV promoter followedby a polylinker region, a SV40 intron and polyadenylation site. A DNAfragment encoding the full length human ACE-2 and a HA or myc tag fusedin frame to its 3′ end is then cloned into the polylinker region of the.The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein as previously described (I. Wilson, H. Niman, R.Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell 37, 767).The infusion of HA tag to ACE-2 allows easy detection of the recombinantprotein with an antibody that recognizes the HA epitope.

For expression of the recombinant ACE-2, COS cells are transfected withthe expression vector by DEAE-DEXTRAN method. (J. Sambrook, E. Fritsch,T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold SpringLaboratory Press, (1989)). The expression of the ACE-2 —HA protein canbe detected by radiolabelling and immunoprecipitation with an anti-HAantibody. (E. Harlow, D. Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory Press, (1988)). For this, transfected cells arelabelled with ³⁵S-cysteine two days post transfection. The cells, oralternatively the culture media (e.g., for the soluble ACE-2) is thencollected and the ACE-2 protein immunoprecipitated with an HA specificmonoclonal antibody. To determine whether full length ACE-2 is amembrane protein, and/or a secreted protein, the cells transfected witha vector encoding the full length ACE-2 protein can be lysed withdetergent (RIPA buffer (150 mM NaCl 1% NP-40, 0.1% SDS, 1% NP-40, 0.5%DOC, 50 mM Tris, pH 7.5). (Wilson, I. et al., Id. 37:767 (1984)).Proteins precipitated can then be analyzed on SDS-PAGE gel. Thus, thepresence of ACE-2 in the cell will be indicative that the full lengthACE-2 can be membrne bound and the presence of ACE-2 in the supernatantwill be indicative that the protein can also be in a soluble form,whether produced as a secreted protein or released by leakage from thecell.

5. Expression of Recombinant ACE-2 in the Baculovirus System

cDNA encoding human ACE-2 protein was cloned in the vector pBac Pak9(Clonetech) and expressed in the baculovirus system as follows. A 10 Lfermentation run was carried out with SF9 cells grown to 1.3×10⁶cells/ml in SF900II SFM (Gibco/Life Technologies), 18 mM L-Glutamine,and 1× antibiotic-antimycotic (from 100× stock Gibco/Life Technologies)at 27° C. Cells were infected at multiplicity of infection of 0.1 withACE2 baculovirus of titer 1.1×10⁹ pfu/ml. At 96 hours post infection,cells were pelleted at 5000 g centrifugation, and the culturesupernatant was collected, frozen and stored at −80° C.

After purification of the protein, as described in the next Example,purified baculovirus expressed human ACE-2 protein was tested for itsactivity and compared to a negative control (from supernatant of mocktransfected cells). For this assay, 10 μl of each fraction from thecolumn was combined with 90 μl of 55 μM MIPH-1 ACE-2 substrate (AnaSpeccustom synthesis;7-Methoxycoumarin-4-yl)acetyl-Ala-Pro-Lys(2,4-Dinitrophenyl)-OH, storedin DMSO at 5 mg/ml) in assay buffer (50 mM Mes pH 6.5(4-Morpholine-ethanesulphonic acid; Boehringer-Mannheim, 223 794); 300mM NaCl; 0.01% Brij 35 (Pierce 28316)), and the reaction monitored for30 minutes. For the most active ACE-2 fractions, a re-assay using 1 in100 diluted fractions was carried out. The results, which are shown inFIG. 4, indicate strong ACE-2 activity in fraction 24.

6. Purification of ACE-2 Protein

This Example describes a method of purification of ACE-2 protein fromsupernatant.

All the steps are carried out at 4° C. Two 1 L. bottles of ACE-2containing supernatant were thawed at 4° C. for 50 hours. The two litersupernatant (pH 6.3) was then adjusted at pH7 with about 6 ml of 2N NaOHadded dropwise while stirring. The pH adjusted supernatant was filteredin a 0.2 μm Nalgene SFCA filter unit. 800 ml of filtered supernatant wasloaded onto a 20.1 ml (16 mmd/100 mml) Pharmacia MonoQ strong anionexchange column at 8 ml/min (239 cm/h). The column had been equilibratedin 50 mM BisTrisPropane/Tris HCl pH7 (Buffer A). After loading thesupernatant, the column was washed to baseline with 40 CV Buffer A.ACE-2 was then eluted with a 0-250 mM NaCl gradient in Buffer A over 20CV while collecting 1 ml fractions. The ACE-2 containing fractions wereresolved by SDS-PAGE analysis and collected in two major pools based onminor differences in purity. A second 800 ml load was completed and twomajor pools were made based on chromatographic simililarity to the firstrun. The four elution pools were dialyzed into 10 mM HEPES pH 7; 15 mMNaCl overnight. After quantitating protein yield based on Bradford assayand A280, followed by comparison of activity relative to a referencesample (071399) generated during assay development, all four pools werepooled together. The purified ACE-2 was then concentrated in a FiltronOmegacell unit with a 10K MWCO membrane. Concentrated ACE was 0.22 μmfiltered in a Millex-GV 25 mm syringe filter in a laminar hood and finalconcentration was determined by Bradford assay.

The purified ACE-2 protein was then used in a high throughput screeningassay described below.

7. Angiotensin I, Neurotensin (1-13) and des-Arg Bradykinin (1-8) areSubstrates of ACE-2

This Example demonstrates that angiotensin I, Neurotensin (1-13) anddes-Arg Bradykinin (1-8) are substrates of ACE-2 and that ACE-2 cleavesthe C-terminal amino acid from each of these peptides. In particular,ACE-2 cleaves Angiotensin I into Ang. (1-9) having the amino acidsequence DRVYIHPFH (SEQ ID NO:16).

The effect of ACE-2 on these peptides was tested by incubating mixturescontaining buffer (10 mM Tris, pH 7) and 5 μL of angiotensin I(DRVYIHPFHL; SEQ ID NO:15) (15 pmol/μL obtained from Sigma-Aldrich Corp.(St. Louis, Mo.), 5 μL Neurotensin, or 5 μL des-Arg Bradykinin (1-8) at15 pmol/μL made by standard procedures with or without 10 μL ACE-2, for30 minutes at 37° C. The human ACE-2 protein used in these assays wasproduced from a cDNA in which a stop codon was inserted after the serineimmediately preceding the transmembrane domain. The protein was purifiedas described supra. A stock solution of the ACE-2 protein was kept in 10mM HEPES (Sigma), 15 nM NaCl (Sigma) stored as aliquots at −70° C.

After the enzymatic reaction, 1 μL samples were removed from eachreaction microtube and the enzymatic reaction was quenched by theaddition of 1 μL of a low-pH MALDI matrix compound, 10 g/L α-cyano-4hydroxycinnamic acid (α-CHCA) in a 1:1 mixture of acetonitrile and water(MALDI matrix solution). 1 μL of each of the resulting quenched reactionmixtures was applied to the surface of a MALDI plate. The plate was thenair-dried and inserted into the sample introduction port of a VoyagerElite Biospectrometry MALDI time-of-flight (TOF) mass spectrometer(PerSeptive Biosystems, Inc., Framingham, Mass.). The resulting signalwas digitized at 1 GHz frequency and accumulated for 64 scans.

Mass spectrometry data for the ACE-2 hydrolysis of angiotensin I (1-10)to angiotensin (1-9) is shown in FIG. 5B, whereas mass spectrometry dateof angiotensin in the absence of ACE-2 is shown in FIG. 5A. FIG. 5Ashows that no hydrolysis occurs in the absence of ACE-2 in the reaction.Similarly, FIGS. 6A and 6B show that ACE-2 hydrolyzes Neurotensin (1-13)into Neurotensin (1-12) (FIG. 6B). No conversion occurs in the absenceof ACE-2 (FIG. 6A). The mass spectrometry data for des-Arg bradykinin(1-8) to des-Arg bradykinin (1-7) is shown in FIG. 7B. No conversion ofbradykinin occurs in the absence of ACE-2 (FIG. 7A).

Thus, this Example demonstrates that ACE-2 is a carboxypeptidase thataccepts a variety of P1′ amino acids with a free carboxylic acid group.

8. Kinetensin is a Substrate of ACE-2

This Example demonstrates that kinetensin is a substrate of ACE-2 andthat ACE-2 cleaves the C-terminal leucine residue from kinetensin.

Kinetensin (IARRHPYFL; SEQ ID NO:23) obtained from Sigma-Aldrich Corp.(St. Louis, Mo.) was incubated at 15 pmol/μL with ACE-2 produced in CHOcells as described above, and the reaction mixture was then subjected tomass spectrometry, also as described above. The results indicate thatACE-2 cleaves the C-terminal leucine residue from kinetensin, thusproducing an 8-mer peptide having the amino acid sequence IARRHPYF (SEQID NO:24).

9. Effect of ACE-2 on Other Peptides

Mass spectroscopy studies showed that ACE-2 cleaves the terminal aminoacid of a peptide only if it contains the free carboxylic acid group.Two commercially available, peptide based substrates Mca-YVADAPK(Dnp), asubstrate for caspase I (Bachem M-2195) and Mca-PLGP-[D-Lys](Dnp), asubstrate for thimet oligopeptidase (Bachem M-2270) with free carboxylicacid groups were used. These types of substrate are known asintramolecularly quenched fluorescent substrates. The presence of theDnp substituted onto the terminal lysine causes quenching of the Mcafluor that is relieved only when an intervening bond is cleaved. WhenMca-YVADAPK(Dnp) was tested as a substrate for ACE-2, an increase offluorescence was measured. No such increase in fluorescence was seenwith Mca-PLGP-[D-Lys](Dnp). It was inferred from these data that thecleavage of Mca-YVADAPK(Dnp) occurred between the proline and lysineresidues and required the lysine to be in the L configuration. The siteof peptide cleavage was confirmed by mass spectroscopy (see FIG. 10A)

Following the discovery that Mca-YVADAPK(Dnp) is an ACE-2 substrate, ashortened form was custom synthesised by AnaSpec (San Jose, Calif., USA)(see FIG. 10B). This substrate, MIPH-1, was found to have higherturnover and lower background fluorescence.

To determine whether ACE-2 is capable of cleaving other peptides, ACE-2produced in CHO cells, alone or with testicular ACE produced in CHOcells, was incubated as described above in 5 μL reactions containing 15pmol/μL of each of the following peptides: angiotensin II (=angiotensin(1-8)), angiotensin (1-7), angiotensin (1-9), angiotensin (2-10),luteinizing hormone releasing hormone (LHRH), des-Gly¹⁰-LHRH Ethylamide,LHRH fragment 4-10, oxytocin, Arg⁸-Vasopressin, litorin, eledoisin,ranatensin, bombesin, renin substrate tetradecapeptide, andadrenocorticotropic hormone fragment 18-39. These peptides were obtainedfrom-Aldrich Corp. (St. Louis, Mo.). MS spectrum of the reactions wereobtained as described in the previous Example. The results showed thatACE-2 had no effect on any of these peptides, alone or in the presenceof testicular ACE.

10. Human ACE-2 Variants

This Example describes variants, e.g., polymorphic variants, of thehuman ACE-2 gene which were found by performing single strandconformation polymorphism (SSCP) studies of the ACE-2 gene in DNA of 96individuals from a randomly ascertained U.S. Caucasian population.

Prior to analyzing ACE-2 variants, the genomic structure of human ACE-2was elucidated. The coding region of the human ACE-2 is comprised in 18exons, the first exon containing the ATG and the 18th exon containingthe TAG codon. The exon/intron borders are shown on FIG. 1. Humangenomic ACE-2 DNA sequence is set forth in GenBank Accession No.AC003669.

PCR was performed with the primer pairs listed in Table III, using 20 ngof template genomic DNA in a final volume of 15 μl. The PCR reactionswere performed by mixing 5 μl template DNA 10 ng/μl; 1.5 μl 10× PerkinElmer PCR Buffer; 1.2 μl Pharmacia dNTP mix 2.5 mM; 1.15 μl Forwardprimer 6.6 μM; 1.15 μl Reverse primer 6.6 μM; 5 μl Gibco/BRL PlatinumTaq 0.05 U/μl (Hot Start); by heating the reaction at 95° C. for 10′;conducting 35 cycles of [94° C. for 40″; 57° C. for 40″; 72° C. for 40″]and terminating by incubating the reactions at 72° C. for 5′. Then, 3.5μl of PCR reaction was added to 4.5 μl SSCP buffer (95% Formamide; 0.1%Bromophenol Blue; 0.1% Xylene Cyanol; 20 mM EDTA). Each amplicon wasdenatured at 97° C. for 10 seconds, immediately chilled on ice, and 8 μlare loaded onto and separated on a nondenaturing, 10% polyacrylamide(39: 1) SSCP gel that was run at 12 volts/gel for 5-6 hours at 4° C.Amplicons with variant SSCP bands along with several non variant sampleswere sequenced on an ABI 377. ABI traces from both strands of eachamplicon were compared to identify nucleotide differences between SSCPvariant and non variant samples. TABLE III PCR Primers Used in SSCPAnalysis of Human ACE-2 ace2e1a/b ace2e1a TTCCCTTTTCAGTTTCACGGGCAG (SEQID NO:30) ace2e1b TCTTCCTGGCTCCTTCTCAGC (SEQ ID NO:31) ace2e1c/d ace2e1cTCTTGGCCTGTTCCTCAATGGTG (SEQ ID NO:32) ace2e1d AGCGCCCAACCCAAGTTCAAAG(SEQ ID NO:33) ace2e2a1/b ace2e2a1 ATGGACACCTTACCTAGGCATAGAG (SEQ IDNO:34) ace2e2b ATCTCACAGTCAAGCTTCAGCTGC (SEQ ID NO:35) ace2e2c/d ace2e2cTGCTCTTGTCTTCTGAGAGCACTG (SEQ ID NO:36) ace2e2dTCTGTTCTATCTCTTCAAGCAATGCC (SEQ ID NO:37) ace2e3a/b ace2e3aCATCTATGTGTTGAAACACACATATCTGC (SEQ ID NO:38) ace2e3bAGGATATCTTTATATTAGCATTCTCTTCAGC (SEQ ID NO:39) ace2e4a/b ace2e4aTAATGCAGAAGAAATAGCCCCGTGG (SEQ ID NO:40) ace2e4bTTGTGTGCTTTGGGATAACAGGTTTG (SEQ ID NO:41) ace2e5a/b ace2e5aATGTGTTAAGAATGAGCCAGAATGCC (SEQ ID NO:42) ace2e5bCTCTTTCTTTCCCTTATGTTCTTCCC (SEQ ID NO:43) ace2e6a/b ace2e6aGCGATTTCTACAATGTTACTAACCAC (SEQ ID NO:44) ace2e6bGTGGAATGGAAATTAGAATTGGTTAC (SEQ ID NO:45) ace2e7a/b ace2e7aCTGCTTTTCCATGAAACTATAGCTAC (SEQ ID NO:46) ace2e7bGGTGATATGTGGGGTAGATTTTGGA (SEQ ID NO:47) ace2e7c/d ace2e7cGGTCCACCATTGCATCAGTAACAT (SEQ ID NO:48) ace2e7dCCAACACTAGGAATTACTAACAGCTT (SEQ ID NO:49) ace2e8a/b ace2e8aCCTGCCTCTGTTGTCTCCCATTTA (SEQ ID NO:50) ace2e8bGAAAATTCCATGCTAACGGACCCAG (SEQ ID NO:51) ace2e8c/d ace2e8cTGGGATGGCAGACTGCTTTCTGAA (SEQ ID NO:52) ace2e8dCGGTGCCTGGCTTATTTAATTTAAGA (SEQ ID NO:53) ace2e9a/b ace2e9aCTCATACCTCATACCTTATGTGGCAA (SEQ ID NO:54) ace2e9bGGCATATGCTGCACAACCTTTTC (SEQ ID NO:55) ace2e9c/d ace2e9cCCCAACAGCTTCATGGAATCCTTCA (SEQ ID NO:56) ace2e9dCCCATACAACTCCACTGTAATGGTT (SEQ ID NO:57) ace2e10a/b ace2e10aCGCCAGTCAAATGCTTTTAAATACAC (SEQ ID NO:58) ace2e10bCATCCACTGTCATCTTCATCGTAAT (SEQ ID NO:59) ace2e11a/b ace2e11aGTTATTAGCACAGCTGTCCACAAAC (SEQ ID NO:60) ace2e11bGATGAAACTGCACTAGTTATGCCC (SEQ ID NO:61) ace2e12a/b ace2e12aCTAGGCATGGAAATGAGTAATACTG (SEQ ID NO:62) ace2e12bGGTTACTTGGGCTCCAGATTTAAAT (SEQ ID NO:63) ace2e13a/b ace2e13aCTGTGTCACAAGTCCTCATGAGACT (SEQ ID NO:64) ace2e13bTGTACATCTGGAACCCCTCAAAAG (SEQ ID NO:65) ace2e14a/b ace2e14aGAACCACATGGCCTCTCTTCTTTC (SEQ ID NO:66) ace2e14bCAGTTACCCCTGTCTCATCATTTCT (SEQ ID NO:67) ace2e15a/b ace2e15aCAGAGTATCTCCTCAGACTCAAGA (SEQ ID NO:68) ace2e15bGGTCACTGACTTAATGAATAGCAAG (SEQ ID NO:69) ace2e16a/b ace2e16aGGCACACAGGAAGAACACACAAAAT (SEQ ID NO:70) ace2e16bCTCTGTGCCACAAGTGAAGATGT (SEQ ID NO:71) ace2e17a/b1 ace2e17aGGTCTATACAATCTACCACTTACTG (SEQ ID NO:72) ace2e17b1AGCCAACACTTGGACCTCCTAAC (SEQ ID NO:73) ace2e17c/d ace2e17cGTGAAGATCAGGATGACAATGCC (SEQ ID NO:74) ace2e17dGCTCTATTATATCCTTTCAGGAACA (SEQ ID NO:75) ace2e18a/b ace2e18aCCCCAGACACTCAGATGATAACTT (SEQ ID NO:76) ace2e18bCAGAGCATGCCTGATAGAAACTCA (SEQ ID NO:77) ace2e18c/d ace2e18cTACCCACTTCAGAGGGTGAACAT (SEQ ID NO:78) ace2e18dGTCAAGGATGACATGCTTTCTTCAC (SEQ ID NO:79) ace2e18e/f ace2e18eCATGATCGATTCCAAACATCACTGT (SEQ ID NO:80) ace2e18fCTGTCTCTGGATTTGACTTCTGTTC (SEQ ID NO:81) ace2e18g/h ace2e18gGCCTGTGAGACCAAATACACACTTT (SEQ ID NO:82) ace2e18hCACTGATGATGTTCAGACCTCCTTT (SEQ ID NO:83) ace2e18i/j ace2e18iCTTGGCCATGTTGTCTTTGGACAA (SEQ ID NO:84) ace2e18jCTCCTTAACACAGATTCCCCTGAA (SEQ ID NO:85)

Five polymorphisms were uncovered in the Caucasian population (FIG. 9and FIG. 13) and three polymorphisms were uncovered in the Asianpopulation (FIG. 13).

The first polymorphism (or variation) in the Caucasian population is a Gto T change in intron 3, which can be detected using primers 3a/3b(ace2e3a and ace2e3b). The sequence encompassing the polymorphism is: 5′TTGAACCAGGTAgGCTACTAATTTT3′ (SEQ ID NO:86; the variant nucleotide isindicated in lower case). The corresponding variant sequence is 5′TTGAACCAGGTAtGCTACTAATTTT3′ (SEQ ID NO:87).

The second polymorphism in the Caucasian population is a 5 bp insertion(CTTAT) in intron 9, which can be detected with primers 9a/9b (ace2e9aand ace2e9b). The sequence encompassing the polymorphism is: 5′GGTATTTATTATGTAGGAAATA3′ (SEQ ID NO:88). The corresponding variantsequence is 5′ GGTATTTATTATcttatGTAGGAAATA3′ (SEQ ID NO:89; the variantnucleotides are indicated in lower case).

The third polymorphism in the Caucasian population is a T to G change inintron 14, which can be detected with primers 14a/14b (ace2e14a andace2e14b). The sequence encompassing the polymorphism is: 5′TGAATTGATTATTtTTGAGTGCACAG3′ (SEQ ID NO:90; the variant nucleotide isindicated in lower case). The corresponding variant sequence is 5′TGAATTGATTATTgTTGAGTGCACAG3′ (SEQ ID NO:91).

The fourth polymorphism in the Caucasian population is an A to G changeat residue 2239 in exon 17, which can be detected with primers 17c/d(ace2e17c and ace2e 17d), and which results in a change of theasparagine at amino acid 720 to an aspartic acid. The sequenceencompassing the polymorphism has the sequence: 5′CGTCTGAATGACaACAGCCTAGA G3′ (SEQ ID NO:92; the variant nucleotide isindicated in lower case). The corresponding variant sequence is 5′CGTCTGAATGACgACAGCCTAGAG3′ (SEQ ID NO:93).

The fifth polymorphism in the Caucasian population is a G to T change atresidue 2834 in the 3′ untranslated region in exon 18, which can bedetected with primers 18e/f (ace2e18e/ace2e18f). The sequenceencompassing the polymorphism has the sequence: 5′AGTTGAAAACAAgGATATATCATTGG3′ (SEQ ID NO:94; the variant nucleotide isindicated in lower case). The corresponding variant sequence is 5′AGTTGAAAAC AAtGATATATCATTGG3′ (SEQ ID NO:95).

The first polymorphism (or variation) in the Asian population is an A toG change in the 5′ untranslated region, which can be detected usingprimers 1c/1d (ace2e1c and ace2e1d). The sequence encompassing thepolymorphism is: 5′ CTAGGGAAAGTCaTTCAGTGGATGTG3′ (SEQ ID NO:96); thevariant nucleotide is indicated in lower case). The correspondingvariant sequence is 5′ CTAGGGAAAGTCgTTCAGTGGATGTG 3′ (SEQ ID NO:97).

The second polymorphism (or variation) in the Asian population is a A toG change in intron 3, which can be detected using primers 3a/3b (ace2e3aand ace2e3b). The sequence encompassing the polymorphism is: 5′TTGAACCAGGTAaGCTACTAATTT 3′ (SEQ ID NO:98); the variant nucleotide isindicated in lower case). The corresponding variant sequence is 5′TTGAACCAGGTAgGCTACTAATT 3′ (SEQ ID NO:99).

The third polymorphism (or variation) in the Asian population is a G toA change in the 3′ untranslated region in exon 18, which can be detectedwith primers 18c/d (ace2e18c/ace2e18d). The sequence encompassing thepolymorphism has the sequence: 5′ GTTCTCTAACTGTgGAGTGAATGGAAA3′ (SEQ IDNO:100); the variant nucleotide is indicated in lower case). Thecorresponding variant sequence is 5′ GTTCTCTAACTGTaGAGTGAATGGAAA 3′ (SEQID NO:101). MMM

5.11. ACE-2 Transgenic Mice

This Example describes the generation of transgenic mice exp ressinghuman ACE-2 cDNA.

The transgenic mice were created by injecting a nucleic acid comprisingthe full length human ACE-2 cDNA (3396 base pairs) under the control ofthe 5.5 kb α-myosin heavy chain into pronuclei of FVB mice according towell known methods. Under control of this promoter, the ACE-2 protein isexpressed in cardiac myocytes. Ten male founders were bred with wildtype females and 70% demonstrated germ line transmission in the F1generation.

Northern analysis of transgenic hearts demonstrated the presence of thefull length transcript as well as the presence of a smaller, moreabundant message. Following RT-PCR of this smaller transcript, the firstATG encoded the longest open reading frame (ORF) which was in frame withACE-2 sequence. Western analysis with an anti-peptide antibody specificfor this region demonstrated the presence of only the full length ACE-2protein and not the alternately spliced form. Western analysis alsodemonstrated the presence of the human ACE-2 protein in transgenicmurine hearts but not in lung, liver, kidney or spleen.

The presence of ACE-2 in the serum of the transgenic and wildtypeanimals was tested by combining 5 μl of serum of the mice and 45 μl of55 μM ACE-2 substrate (see above) in ACE-2 buffer and measuring the rateof proteolytic degradation of the substrate by measuring the productionof fluorescence (in flurorescence units) per second for 30 minutes atroom temperature at a gain setting of 10. The average rate offluoresence units per second (FU/sec) correlates directly with theamount of ACE-2 in the serum. As a control for the specificity of ACE-2,a standard carboxypeptidase assay was performed (Holmquist and Riordan,Carboxypeptidase A, pp44-60, Peptidase and their Inhibitors in Method ofEnzymatic Analysis (1984). The results are shown in Table IV. TABLE IVACE-2 Transgenic versus Wildtype Serum Activity Line and Line and mouseType Av. Rate mouse Type Av. Rate a3320 FU/sec a3503 FU/sec cardiacbleed a3617-2.2f wt 0.103 99-8082.24 wt 0.088 a3617-2.3f wt 0.11399-8082.27 wt 0.106 a3614-2.1f tg 0.234 99-8082.25 tg 0.622 a3615-2.1ftg 0.191 99-8082.26 tg 0.412 a3617-2.1m wt 0.148 a3617-5.2m wt 0.130a3614-2.1m tg 0.211 a3617-5.1m tg 0.296

Enzymatic cleavage of ACE-2 assay substrate was approximately 5 foldhigher in line 2 serum and 2 fold higher in line 1 serum relative tolittermate controls. This indicates that there is circulating ACE-2 inthe blood of these transgenic animals. These animals can be used foranalyzing any potential inhibitors of ACE-2.

Mice from line 1 were also analyzed for weight and length. These micewere slightly shorter in length than their littermate counterparts andwere significantly lower in body weight. Weight to length ratios weresignificantly lower in tansgenics than littermate controls, suggestingthat transgenic mice may be leaner than normal.

Blood chemistries (glucose, blood urea nitrogen, creatinine, totalbilirubin, and a number of cardiac markers including alanine aminotransferase, asparagine amino transferase, creatine phosphokinase,sodium, potassium, chloride, calcium, IP, and magnesium) from transgenicline 1 animals and from littermate controls were examined. A significantincrease in chloride levels were found in transgenics (114.8 mmol/)versus controls (112.0 mmol/l) (students T-test p=0.0009). The liverenzymes ALT and AST also were higher in transgenics than in controls(p=0.024 and p=0.084, respectively).

Sudden death has been observed in both lines. In line 2 mice, deathsstart to occur at 4 weeks of age with only 40% of the trarsgenic micesurviving at day 45 (100% of wild type mice survive at day 45). Deathsstart to occur in line 1 after 3 months of age. The mice from line 1have been evaluated for functional cardiac changes. In these mice,cardiac contractility was found to be normal, however, their bloodpressure was reduced relative to wild type littermates.

Phenotypic analysis of these mice was undertaken in 2 lines of thesetransgenic mice (designated line 1 and line 2). Histological analysis ofhearts from both of these lines shows subtle mycoyte vaculolization,focal ischemic changes and myocyte loss, splaying of the myofibrils, andfocal areas of hemorrhage. The spleens of several animals showed signsof congestion. Livers have chronic passive congestion and centrilobularhepatocyte necrosis and hypotensive changes. The brains of severalanimals showed mild to moderate selective neuronal necrosis,particularly in the CA1, 2, and 3 subsectors onf the hippocampus and inthe frontoparietal cortex. These changes are consistent with hypotensiveand global ischemic insults. In older transgenic mice, the ischemicchanges in the myocardium (myocyte contraction bands; increase in eosinuptake by injured myocytes) appears moire diffuse and prominent.

To eliminate the possibility that the smaller transcript in line 1 and 2transgenics caused any observable phenotype, transgenic mice werecreated using the human ACE-2 cDNA, in which the cryptic splice acceptorsite was removed by changing bases 2167-2172, from CCTAGA to CCGCGC.These changes were made such that the mRNA would still encode theidentical full-length ACE-2 protein, however, the bases in the RNA wouldnot be recognized as a splice acceptor site. This ACE-2 cDNA was thenoperably linked to the myosin heavy chain promoter, introduced intopronuclei, to obtain transgenic mice. The resulting transgenic mice,designated as line 3, contained only the correct size transcript asdetermined by Northern blots of mRNA from their heart. Results indicatethat mice from this group of animals also die at a very young age,demonstrating that the smaller transcript in lines 1 and 2 was not thecause of early death.

ACE-2 transgenic mice were also subjected to surface electrocardiograms(ECG) and Holter monitoring. For surface EGC recordings, mice wereanesthetized with 2.5% Avertin and 27 G needles electrodes (positive,negative, and ground) were subdermally placed onto the left hind limb,tail and right forepaw, respectively. The leads were connected to adifferential amplifier and the data was digitized at 4 K/s and analyzedusing the Chart analysis software. ACE-2 transgenic mice exhibitedcomplete atrioventricular block as measured by independent P waves andORS complexes (FIGS. 11A-C). In addition these mice exhibited a higherpreponderance of premature ventricular beats (PVB) (FIGS. 11A-C).

In order to determine whether the high incidence of mortality observedin these transgenic mice was due to sudden cardiac death, these micewere telemetered (using a Holter monitor, described in, for example,Gehrmann J. and Beroul C. I. (2000) J. Cardiovascular Electrophysiology11:354-68) and continuous conscious ECG were recorded. In one of thetransgenic telemetered mice which exhibited the usual pattern ofcomplete atrioventricular block there was recorded evidence of nonsustained ventricular tachycardia which progressed into ventricularfibrillation and ultimately resulted in asystole and death, suggestingthe appearance of sudden cardiac death (FIG. 12).

12. ACE-2 Knock-Out Mice

This example describes the generation of mice having one or both ACE-2genes disrupted by the insertion of a neo/ura cassette in the siteencoding the active site of the enzyme.

A BAC clone, BAC145d21, containing a genomic fragment that includes theexon encoding the active site of the ACE-2 enzyme was isolated byscreening an RPCI-22 mouse BAC library with a 1.8 Kb EcoRI fragment fromthe human ACE-2 cDNA. A 5.4 kb murine genomic ACE-2 fragment containingthe exon including the active site of ACE-2 was subcloned into the yeastshuttle vector YCplac22.

A random sheared library was prepared from BAC145d21 for sequencing.Base perfect sequence was obtained from the region flanking andincluding the exon encoding the active site. A PCR primer containing 45bp of murine ACE-2 sequence corresponding to the region flanking theactive site on the 5′ end was prepared. The 3′ end of this primercontained 21-24 bp of sequence corresponding to the neo/ura cassette inthe plasmid pRAY-1 and desigated as the forward primer. The reverseprimer was similarly designed with ACE-2 sequences downstream of theactive site and the 3′ end of the neo/ura cassette. The PCR productobtained from amplification of mouse DNA using these two primerscontained the neo/ura cassette flanked by ACE-2 sequence. This PCRproduct was then co-transformed with the YCplac22-ACE-2 construct intothe yeast strain YPH501 to obtain homologously recombinated DNA, i.e.,YCplac-ACE-2 having an neo/ura cassette in the site encoding the activesite of the ACE-2 enzyme. Replacement of the exon encoding the activesite with the neo/ura cassette was confirmed by restriction digestion oftransformants. Integrity of the neo/ura cassette in the knockoutconstruct was confirmed by transfection into CHOK1 cells and selectionwith G418.

The ACE-2 knockout construct was linearized and electroporated into EScells. ES cells having undergone homologous recombination were selectedby culture of the ES cells in the presence of G418. Clones were screenedby Southern blot using 5′ and 3′ flanking probes to confirmrecombination. ES cell clones having undergone homologous recombinationwere then injected into blastocyts and transferred to pseudopregantfemale mice for generating chimeric mice. Male chimeras were mated withC57B16 females to obtain germline transmission of knockout. Ten pups ofthe correct coat color (agouti) indicating germline transmission, wereobtained. Since the ACE-2 gene has been mapped to the X chromosome (seebelow), heterozygous females were generated. These can be mated bystandard crossings to obtain hemizygous males, heterozygous females, andhomozygous females for the widtype or the knock-out allele.

13. The Human ACE-2 Gene is Located on the X Chromosome at p21-22

This example demonstrates that the human and mouse ACE-2 genes arelocated on the X chromosome.

Chromosome localization of the human ACE-2 gene was performed byamplifying the 93 DNAs from the Genebridge 4 Radiation Hybrid Panel induplicate. The primers were chosen based on their ability to hybridizeto the DNA of a human cell line, but not to that of a control hamstercell line, and consisted of the following sequences: 5′GGATCACTTGTAAGGACAGTGCC 3′ (forward primer; SEQ ID NO:102) and 5′GATCGATTCCAAACATCACTGTAGGC 3′ (reverse primer; SEQ ID NO:103).Amplification results in a 169 bp DNA fragment.

The PCR reactions were performed by mixing 5 μl Template DNA 10 ng/μl;1.5 μl 10× Perkin Elmer PCR Buffer; 1.2 μl Pharmacia dNTP mix 2.5 mM;1.15 μl Forward primer 6.6 μM; 1.15 μl Reverse primer 6.6 μM; 5 μlGibco/BRL Platinum Taq 0.05 U/μl (Hot Start); by heating the reaction at95° C. for 10′; conducting 35 cycles of (94° C. for 40″; 55° C. for 40″;72° C. for 40″] and terminating by incubating the reactions at 72° C.for 5′. The PCR products were run on 2% agarose gels, post-stained withSYBR Gold (1:10,000 dilution in 1×TBE), and scanned on a MolecularDynamics 595 Fluorimager.

The results indicated that ACE2 maps to the p-arm of the human Xchromosome, 37.1 cR₃₀₀₀ centromeric to the Whitehead Institute frameworkmarker DXS1223, and 42.0 cR₃₀₀₀ telomeric of the Whitehead frameworkmarker DXS1061. LOD scores for linkage were 6.6 for DXS1223 and 6.1 forDXS1061. This region corresponds to the cytogenetic location Xp21-22,which is syntenic to the mouse X chromosome.

14. The Mouse ACE-2 Gene is Located on the X Chromosome

Chromosome localization of the mouse ACE-2 gene was performed asfollows. PCR primers were designed from conserved regions flanking a CAmicrosatellite of 28 repeat units (56 bp long) in mouse genomic ACE-2DNA. The forward primer had the sequence 5′ ATTGACCATTGTTGGAACACTACCG 3′(SEQ ID NO:104) and the reverse primer had the sequence 5′GTGTGTTAGCCCCTCCTGGC 3′ (SEQ ID NO: 105). These primers were used toamplify a 321 bp PCR product from C57BL/6J DNA and a smaller PCR productfrom wild type derived Mus spretus strain SPRET/Ei DNA. PCR reactionswere performed by combining 6 μl Template DNA 10 ng/μl; 1.4 μl 10×Perkin Elmer PCR Buffer; 1.12 μl dNTPs 2.5 mM; 1.05 μl Forward primer6.6 μM; 1.05 μl Reverse primer 6.6 μM; 0.38 μl H₂O; 3 μl AmpliTaq 0.05U/μl (Hot Start), and incubating the reaction mixture for 35 cycles of[94° C. for 40″; 55° C. for 50″; 72° C. for 30″]. The products were runon a nondenaturing 8% polyacrylamide gel at 45 W at room temperature for3 hrs for size determination (SSLP analysis). Gels were stained,postelectrophoresis, with SYBR Gold and scanned on a Molecular Dynamics595 Fluorimager.

The genetic segregation of the M. spretus allele was followed in 186progeny of a (C57BL/6J×M. spretus)×C57BL/6J mapping panel by SSLP(Simple Sequence Length Polymorphism) analysis. The segregation patternof the M. spretus allele was compared with the segregation pattern of394 other genetic loci that have been mapped in this backcross panel. Byminimizing the number of multiple crossovers between ACE-2 and othermarkers, it was determined that ACE maps to the murine X chromosome,approximately 29.71±3.45 cM distal to the marker DXMIT8 and 7.39±1.97 cMproximal of the marker DXMIT12.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1.-20. (canceled)
 21. A method for identifying an ACE-2 therapeutic,comprising contacting an ACE-2 polypeptide with a test compound anddetermining an ACE-2 bioactivity, such that a difference in thebioactivity of the ACE-2 polypeptide contacted with the test compoundrelative to an ACE-2 polypeptide that was not contacted with the testcompound, indicates that the test compound is an ACE-2 therapeutic. 22.The method of claim 20, wherein the ACE-2 bioactivity includes bindingof an ACE-2 polypeptide to a binding partner, and the method comprises(i) combining an ACE-2 polypeptide and an ACE-2 binding partner, and atest compound under conditions wherein, but for the test compound, theACE-2 polypeptide and ACE-2 binding partner form a complex; and (ii)detecting the formation of an ACE-2 polypeptide/ACE-2 binding partnercomplex, such that a difference in the formation of an ACE-2polypeptide/ACE-2 binding partner complex in the presence of a testcompound relative to the absence of the test compound indicatives thatthe test compound is an ACE-2 therapeutic.
 23. The method of claim 21for identifying an ACE-2 antagonist, comprising (i) combining an ACE-2polypeptide and an ACE-2 binding partner, and a test compound underconditions wherein, but for the test compound, the ACE-2 polypeptide andACE-2 binding partner form a complex; and (ii) detecting the formationof an ACE-2 polypeptide/ACE-2 binding partner complex, such that areduction in the formation of an ACE-2 polypeptide/ACE-2 binding partnercomplex in the presence of a test compound relative to the absence ofthe test compound indicatives that the test compound is an ACE-2antagonist.
 24. The method of claim 21, wherein the ACE-2 bindingpartner is a target peptide or analog thereof.
 25. The method of claim23, wherein the binding partner is angiotensin I or a portion thereof oran analog thereof sufficient for binding to an ACE-2 polypeptide. 26.The method of claim 23, wherein the binding partner is angiotensin I ora portion thereof or an analog thereof sufficient for binding to, andbeing hydrolyzed by, an ACE-2 polypeptide.
 27. The method of claim 23,wherein the binding partner is kinetensin or a portion thereof or ananalog thereof sufficient for binding to an ACE-2 polypeptide.
 28. Themethod of claim 26, wherein the binding partner is kinetensin or aportion thereof or an analog thereof sufficient for binding to, andbeing hydrolyzed by, an ACE-2 polypeptide.
 29. (canceled)
 30. (canceled)31. The method of claim 20, wherein the ACE-2 bioactivity includescleavage of a target peptide by an ACE-2 polypeptide, and the methodcomprises (i) combining into a reaction mixture an ACE-2 polypeptide andan ACE-2 target peptide or analog thereof, and a test compound underconditions wherein, but for the test compound, the ACE-2 polypeptidecleaves one or more amino acids from the ACE-2 target peptide or analogthereof, thereby producing an ACE-2 target peptide conversion product;and (ii) detecting in the reaction mixture the presence of at least oneof the ACE-2 target peptide or analog thereof, the ACE-2 target peptideconversion product, and the one or more amino acids, such that adifference in the level of at least one of the ACE-2 target peptide oranalog thereof, the ACE-2 target peptide conversion product, and the oneor more amino acids in the reaction mixture containing the test compoundrelative to a reaction mixture that does not contain the test compoundindicatives that the test compound is an ACE-2 therapeutic.
 32. Themethod of claim 30, for identifying an ACE-2 antagonist, comprising (i)combining into a reaction mixture an ACE-2 polypeptide and an ACE-2target peptide or analog thereof, and a test compound under conditionswherein, but for the test compound, the ACE-2 polypeptide cleaves one ormore amino acids from the ACE-2 target peptide or analog thereof,thereby producing an ACE-2 target peptide conversion product; and (ii)detecting in the reaction mixture the presence of at least one of theACE-2 target peptide or analog thereof, the ACE-2 target peptideconversion product, and the one or more amino acids, such that a lowerlevel of at least one of the ACE-2 target peptide conversion product andthe one or more amino acids produced, or a higher level of the ACE-2target peptide or analog thereof, in the reaction mixture containing thetest compound relative to a reaction mixture that does not contain thetest compound indicatives that the test compound is an ACE-2 antagonist.33. The method of claim 30, wherein the target peptide is angiotensin Ior a portion thereof or an analog thereof sufficient for being cleavedby an ACE-2 polypeptide.
 34. The method of claim 30, wherein the targetpeptide is kinetensin or a portion thereof or analog thereof sufficientfor being cleaved by an ACE-2 polypeptide.
 35. (canceled)
 36. (canceled)37. The method of claim 30, wherein detecting in the reaction mixturethe presence of at least one of the ACE-2 target peptide or analogthereof, the ACE-2 target peptide conversion product, and the one ormore amino acids comprises obtaining a mass spectrum of the reactionmixture or of a part thereof. 38-42. (canceled)
 43. A method foridentifying a substrate of an ACE-2 polypeptide, comprising (i)contacting an ACE-2 polypeptide with a test compound in a reactionmixture under conditions in which the ACE-2 polypeptide is able tocleave a substrate; and (ii) determining the mass spectrum of thereaction mixture of step (a) or a part thereof, such that the presenceof a lower peak characteristic of the test compound in the reactionmixture of step (a) relative to that in a reaction mixture that does notcontain the ACE-2 polypeptide, indicates that the test compound is asubstrate of the ACE-2 polypeptide.
 44. An isolated antibody or antigenbinding fragment thereof which binds a polypeptide selected from thegroup consisting of: a) a polypeptide comprising the amino acid sequenceset forth in SEQ ID NO:2; b) a polypeptide encoded by the nucleic acidhaving ATCC Designation No. 209510; c) a polypeptide consisting of theamino acid sequence set forth in SEQ ID NO:2; and d) a polypeptidecomprising at least 50 consecutive amino acid residues of SEQ ID NO:2and which has at least one bioactivity of an ACE-2 polypeptide; whereinthe bioactivity is selected from the group consisting of: (i) binding toa target peptide; (ii) catalyzing hydrolysis of a target peptide; and(iii) interacting with a metal ion selected from Zn²⁺ Co²⁺, and Mn²⁺.45. The antibody of claim 44, which is a monoclonal or a bispecificantibody or antigen binding fragment thereof.
 46. The antibody of claim44, which is a humanized, chimeric, or single chain antibody or antigenbinding fragment thereof.
 47. The antibody of claim 44, which comprisesa detectable label.
 48. The antibody of claim 44, wherein the antibodyis a blocking antibody which has the ability to inhibit at least onebioactivity of an ACE-2 polypeptide.